Eicosapentaenoic acid (EPA) formulations

ABSTRACT

Provided herein are compositions comprising eicosapentaenoic acid (EPA) and polar lipids (e.g., glycolipids and phospholipids), and which do not contain any docosahexaenoic acid (DHA) or esterified fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/797,802, filed on Mar. 12, 2013, which issued as U.S. Pat. No.9,629,820 on Apr. 25, 2017, which claims the benefit of U.S. ProvisionalAppl. No. 61/745,740, filed on Dec. 24, 2012, which are herebyincorporated herein by reference in their entireties for all purposes.

FIELD

Provided herein are compositions comprising eicosapentaenoic acid (EPA)and polar lipids (e.g., phospholipid and glycolipids), and which do notcontain any docosahexaenoic acid (DHA).

BACKGROUND

The main source of EPA formulations to date is either fish oil or krill.In the case of fish oil, there are several problems: depletion offisheries, a relatively low EPA content, DHA and the other six Omega-3compounds in the mixture, and a variance in EPA content based on naturalvariance within and between species. The presence of the five otherOmega-3 compounds is problematic as they compete with EPA for access toprotein receptors. Due to fisheries depletion, at least one producer offish oil has had its Atlantic menhaden allocation reduced by 20% (on theinternet atnutraingredients-usa.com/Industry/Omega-Protein-s-Atlantic-menhaden-catch-to-be-cut-by-20).

The lower concentration of EPA in the raw fish oil and the presence ofother near molecular weight components results in refining loss. Fishoil does not include any glycolipids. Phospholipids (PL) present in theraw fish oil tend to be removed through degumming steps adapted from theoilseed industry that as specifically designed to remove thesecomponents. Moreover, the transesterification to ethyl esters, one stepalong the most common refinement methods, also tends to destroy thephospholipids. Phospholipids in the final product would be less than 0.5wt %.

With respect to krill oil, some of the same problems apply. Krill(Euphausia superba) naturally occur in the Antarctic. Krill isconsidered by many scientists to be the largest biomass in the world.Antarctic krill is fundamental to the survival of almost every speciesof animal that lives in the Antarctic or sub-Antarctic waters and islandgroups. Krill also contain eight Omega-3 fatty acids. Many of the fattyacids in Krill are nearly the same molecular weight as EPA and,therefore difficult to remove via refining. The other Omega-3s competefor receptors and, thus, decrease the EPA that is present. Krill, too,have a broad variation in the Omega-3 content and are very susceptibleto breakdown of the PLs into FFA by both thermal and enzymatic action.

If everyone in the US and Europe ingested 2 g per day of EPA, a levelthat has been demonstrated to be effective in cardiovascular and mentalhealth, there is not enough fish in the sea to provide a sustainablesupply.

SUMMARY

Provided are EPA formulations with improved bioavailability by virtue ofcontaining increased concentrations of EPA in its more bioavailableforms (e.g., as free fatty acid, as glycolipid conjugate and asphospholipid conjugate), and reduced or eliminated concentrations of EPAin its least bioavailable forms (e.g., as diglyceride or triglycerideconjugate).

Accordingly, in varying embodiments, EPA compositions comprising fromabout 15 wt. % to about 90 wt. % eicosapentaenoic acid (EPA), and about10 wt. % to about 70 wt. % polar lipids, wherein the composition doesnot comprise docosahexaenoic acid (DHA) or esterified EPA, and whereinthe composition is suitable for human consumption. Herein, DHA refers toDHA in any of the lipid forms including free fatty acid, triglyceride,diglyceride, monoglyceride, sphingolipid, phospholipid, and glycolipid.In some embodiments, an EPA compositions are provided comprising thefollowing distribution of EPA by lipid class: about 3 wt. % to about 50wt. % of the EPA is a phospholipid conjugate; about 5 wt. % to about 50wt. % of the EPA is a glycolipid conjugate; about 0 wt. % to about 10wt. % of the EPA is a triglyceride conjugate or a diglyceride conjugate;and about 15 wt. % to about 85 wt. % of the EPA is in free fatty acidform, and wherein the composition is suitable for human consumption. Insome embodiments, the composition comprises about 15 wt. % to about 75wt. % EPA, e.g., about 20 wt. % to about 50 wt. % EPA. In varyingembodiments, the EPA is in one or more forms selected from the groupconsisting of a free fatty acid a phospholipid conjugate, a glycolipidconjugate, a triglyceride conjugate, and a diglyceride conjugate. Insome embodiments, about 0 wt. % to about 10 wt. % of the EPA in thecomposition is a triglyceride conjugate or a diglyceride conjugate,e.g., less than about 0.2 wt. % of the EPA in the composition is atriglyceride conjugate or a diglyceride conjugate. In some embodiments,about 15 wt. % to about 85 wt. % of the EPA in the composition is infree fatty acid form, e.g., at least about 20 wt. %, 25 wt. %, 30 wt. %,35 wt. %, 40 wt. %, 50 wt. % to about 85 wt. % of the EPA in thecomposition is in free fatty acid form. In some embodiments, about 5 wt.% to about 90 wt. % of the EPA in the composition is a polar lipidconjugate, e.g., about 10 wt. % to about 80 wt. % of the EPA in thecomposition is a polar lipid conjugate. In some embodiments, thecomposition comprises at least about 13 wt. % polar lipids, e.g., atleast about 14 wt. %, 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %,20 wt. % polar lipids. In some embodiments, the polar lipids arecomprised of phospholipid conjugates and glycolipid conjugates at a wt.% ratio in the range of about 3:1 to about 1:3. In some embodiments,about 5 wt. % to about 50 wt. % of the EPA in the composition is aglycolipid conjugate. In some embodiments, glycolipid conjugatescomprise one or more of digalactosyldiacylglycerol andmonogalactosyldiacylglycerol. In some embodiments, about 3 wt. % toabout 50 wt. % of the EPA in the composition is a phospholipidconjugate. In some embodiments, the phospholipid conjugates comprise oneor more of phosphatidylcholine, lyso-phosphatidylcholine,phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol.In some embodiments, the phospholipid conjugates comprise one or more ofphosphatidylcholine and phosphatidylglycerol. In some embodiments, theEPA to total omega-3 fatty acids ratio is greater than 90%, e.g.,greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater. Insome embodiments, the composition comprises at least about 13 wt. %polar lipids, e.g., at least about 14 wt. %, 15 wt. %, 16 wt. %, 17 wt.%, 18 wt. %, 19 wt. %, 20 wt. % polar lipids, less than 0.2 wt. %glyceride conjugates and at least about 30 wt. % free fatty acids. Insome embodiments, the composition comprises about 20-50 wt. % EPA, about10-25 wt. % glycolipids, and about 5-25 wt. % phospholipids. In varyingembodiments, the composition comprises chlorophyll a. In varyingembodiments, the composition does not comprise chlorophyll c. In someembodiments, the composition comprises less than about 10.0 wt. %arachidonic acid, e.g., less than about 9.5 wt. %, 9.0 wt. %, 8.5 wt. %,8.0 wt. %, 7.5 wt. %, 7.0 wt. %, 6.5 wt. %, 6.0 wt. %, 5.5 wt. %, 5.0wt. %, 4.5 wt. %, 4.0 wt. %, 3.5 wt. %, 3.0 wt. %, 2.5 wt. %, 2.0 wt. %,1.5 wt. % or 1.0% arachidonic acid or does not comprise arachidonicacid. In some embodiments, the EPA composition does not comprise or issubstantially free of intact cells, cellular components,polynucleotides, and polypeptides. In some embodiments, the compositioncomprises coenzyme Q9 (CoQ9) and/or coenzyme Q10 (CoQ10). In someembodiments, the composition comprises less than about 1 wt. %phytosterols. In some embodiments, the composition comprises less thanabout 2 wt. % carotenoids. In some embodiments, the composition does notcomprise fatty acids selected from the group consisting ofoctadecatetraenoic acid or stearidonic acid (SDA=C18:4ω3),eicosatrienoic acid (ETE=C20:3ω3), eicosatetraenoic acid (ETA=C20:4ω3),heneicosapentaenoic acid or uncosapentaenoic acid (HPA=C21:5ω3), anddocapentaenoic acid (DPA=C22:5ω3). In some embodiments, the compositiondoes not comprise one or more, e.g., two or more, e.g., or all of thecarotenoids selected from the group consisting of astaxanthin,cis-lutein, trans-lutein, cis-zeaxanthin, trans-alpha-crytoxanthin,trans-alpha-carotene, cis-alpha-carotene, cis-lycopene, andtrans-lycopene. In some embodiments, the composition does not compriseone or more phospholipids selected from the group consisting ofN-acyl-phosphatidylethanolamine, lyso-phosphatidylcholine,phosphatidylinositol and phosphatidylethanolamine. In some embodiments,the composition does not comprise sphingolipids.

In some embodiments, the composition comprises the followingdistribution of EPA by lipid class:

-   -   about 3 wt. % to about 50 wt. % of the EPA is a phospholipid        conjugate;    -   about 5 wt. % to about 50 wt. % of the EPA is a glycolipid        conjugate;    -   about 0 wt. % to about 10 wt. % of the EPA is a triglyceride        conjugate or a diglyceride conjugate; and    -   about 15 wt. % to about 85 wt. % of the EPA is in free fatty        acid form.

In some embodiments, the composition comprises:

i) 0 to 5 wt. % C:18 fatty acids;

ii) 0 to 20 wt. % C:16 fatty acids;

iii) 0 to 5 wt. % C:14 fatty acids;

iv) 0 to 0.5 wt. % C:12 fatty acids; and/or

v) 0 to 0.5 wt. % C:10 fatty acids.

In varying embodiments, the composition comprises:

Component wt. % Fatty Acids Capric (10:0) 0.1-0.2 Lauric (12:0) 0.1-0.2Myristic (14:0) 1.0 Palmitic (16:0) 5.3-6.5 Palmitoleic (16:1) 7.0-8.0Hexadecadienoic (16:2) 0.2-0.3 Stearic (18:0) 0.2-0.3 Oleic (18:1ω9) 2.0Oleic (18:1ω7) 0.3-0.4 Linoleic (18:2ω6) 1.2-2.0 Alpha-Linolenic (ALA)(18:3ω3) 0.2 Arachidonic (20:4ω6) 1.0-8.0 Eicosapentaenoic (EPA)(20:5ω3) 19-30 Total Fatty Acid 40-55 Total Omega-3 20-30EPA/Omega-3 >93    Total Omega-6  2-10 Phospholipids Phosphatidylcholine4.7-7.4 Lyso-Phosphatidylcholine 0.3-0.4 Phosphatidylinositol 0.8-1.3Phosphatidylethanolamine 0.5-0.8 Phosphatidylglycerol 1.8-2.8Glycolipids Digalactosyldiacylglycerol 10-17Monogalactosyldiacylglycerol 3-5 Phytonutrients Phytosterols 1.5Chlorophyll 4-5 Total Phospholipids (PL) (wt %)  9-14 Total Glycolipids(GL) (wt %) 13-21 Total PoL (PL + GL) (wt %) 22-35

In a further aspect, provided is a capsule, tablet, solution, syrup, orsuspension suitable for human consumption comprising an EPA compositionas described above and herein. In varying embodiments, the capsule is agel capsule. Further provided is a food, beverage, energy bar, ornutritional supplement comprising an EPA composition as described aboveand herein.

Further provided are methods of preventing, ameliorating, mitigating,delaying progression of and/or treating a disease condition selectedfrom the group consisting of psychiatric disorders, cardiovasculardisease, liver disease; chronic hepatitis; steatosis; liver fibrosis;alcoholism; malnutrition; chronic parenteral nutrition; phospholipiddeficiency; lipid peroxidation; disarrhythmia of cell regeneration;destabilization of cell membranes; menopausal or post-menopausalconditions; cancer; aging; benign prostatic hyperplasia; kidney disease;edema; skin diseases; gastrointestinal diseases; pregnancy toxemia;arthritis; osteoporosis; inflammatory diseases; and neurodegenerativediseases. In some embodiments, the methods comprise administering to asubject in need thereof an effective amount of a composition, capsule,tablet, solution, syrup, suspension, food, beverage, energy bar, ornutritional supplement as described above and herein. In varyingembodiments, administration is orally or transdermally. In someembodiments, the disease condition is a psychiatric disorder selectedfrom the group consisting of depression, unipolar depression, majordepression, depressed mood and/or post-partum depression, bipolardisorder, anxiety, panic and social phobic disorders, mood disorders,schizophrenia, Obsessive Compulsive Disorder (OCD), borderlinepersonality disorder, attention deficit hyperactivity disorder andrelated disorders, and anorexia nervosa. In some embodiments, thedisease condition is a cardiovascular disease selected from the groupconsisting of hypertension, coronary artery disease,hypercholesterolemia, dyslipidaemia, high blood pressure, and peripheralvascular system disease.

Further provided are methods of producing a composition comprising EPA.In some embodiments, the methods comprise:

-   -   a) providing an algal paste;    -   b) extracting lipids from the algal paste with an organic        solvent, thereby substantially isolating a crude algae extract        (CAE) comprising neutral lipids and polar lipids from the        water-soluble components of the paste;    -   c) substantially removing the remaining water-soluble components        from the CAE, thereby yielding a crude algae oil (CAO);    -   d) contacting the CAO with supercritical CO2, wherein the        supercritical CO2 selectively extracts the neutral lipids,        thereby splitting the CAO into a neutral lipid fraction        comprising free fatty acids and a polar lipid fraction        comprising glycolipids and phospholipids;    -   e) isolating C20 free fatty acids from the neutral lipid        fraction, thereby yielding a concentrated EPA free fatty acid        fraction; and    -   f) combining the concentrated EPA free fatty acid fraction        produced in step e) and the polar lipid fraction produced in        step d).

In some embodiments, the methods comprise:

-   -   a) providing an algal paste;    -   b) extracting lipids from the algal paste with an organic        solvent, thereby isolating a crude algae extract (CAE)        comprising neutral lipids and polar lipids from the        water-soluble components of the paste;    -   c) substantially removing the remaining water-soluble components        from the CAE, thereby yielding a crude algae oil (CAO);    -   d) hydrolyzing a first portion of the CAO, thereby releasing        free fatty acids in the portion of CAO;    -   e) fractionating the released free fatty acids according to        chain length, thereby isolating C20 free fatty acids and        yielding a concentrated EPA free fatty acid fraction; and    -   f) combining the concentrated EPA free fatty acid fraction        produced in step e) and a second portion of the CAO produced in        step c).

In some embodiments, the paste is a wet paste. In some embodiments, thealgal cells comprise Nannochloropsis cells. In some embodiments, theNannochloropsis cells are selected from N. oculata, N. oceanica, andmixtures thereof. In some embodiments, the algal cells further compriseNannochloris cells (e.g., less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or 1% of the total cells), e.g., as a typical by-product ofoutdoor cultivation. In varying embodiments, the method does notcomprise an esterification step. In some embodiments, the algal cellsare not subject to mechanical cracking, thermal pretreatment, alkalinetreatment and/or acid treatment. In some embodiments, the cell membranesof the algal cells are not disrupted. In some embodiments, the paste hasnot been subject to drying. In some embodiments, the paste has not beensubject to thermal drying, vacuum drying, ambient temperature drying,and/or freeze drying. In some embodiments, the solvent is selected fromthe group consisting of an ether, a ketone, an alcohol, and mixturesthereof. In some embodiments, the solvent is selected from the groupconsisting of ethanol, isopropyl alcohol, acetone, dimethyl ether, andmixtures thereof. In some embodiments, the organic solvent is selectedfrom the group consisting of absolute ethanol, 190 proof (95 v/v %)ethanol (EtOH), denatured 190 proof ethanol, special denatured alcohols(SDA), acetone and ethanol, isopropyl alcohol, acetone and methanol,methyl ethyl ketone (MEK) and methanol, MEK and ethanol, dimethyl ether,dimethyl ether and methanol, dimethyl ether and ethanol. In someembodiments, the organic solvent is a mixture of dimethyl ether andethanol. In some embodiments, the supercritical CO2 is maintained at apressure in the range from about 100 bar to about 1000 bar, e.g., in therange from about 340 bar to about 700 bar, e.g., in the range from about350 bar to about 690 bar and at a temperature in the range from about35° C. to about 110° C., e.g., in the range from about 40° C. to about110° C., e.g., in the range from 60° C. and 90° C. In some embodiments,the neutral lipid fraction is subject to hydrolysis, thereby releasingfree fatty acids. In some embodiments, a portion of the CAE is subjectto hydrolysis, thereby releasing free fatty acids. In some embodiments,a portion of the CAO is subject to hydrolysis, thereby releasing freefatty acids. In some embodiments, the neutral lipid fraction, the CAE orthe CAO is exposed to heat, alkali and/or acid to effect hydrolysis. Insome embodiments, the C20 free fatty acids are isolated from thereleased free fatty acids by fractionating the free fatty acids over apressure gradient of supercritical CO2, e.g., a stepwise or continuouspressure gradient of supercritical CO2. In varying embodiments, thepressure gradient of supercritical CO2 is from about 172 bar to about345 bar. In varying embodiments, the pressure gradient of supercriticalCO2 is isothermal, e.g.i, is maintained at a constant temperature ofbetween about 50° C. and about 70° C.

In a further aspect, EPA compositions produced by the methods describedabove and herein are provided.

DEFINITIONS

The term “substantially” with respect to isolation, removal orpurification refers to at least about 90% isolated, removed and/orpurified, e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, isolated, removed or purified.

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, andtopical/transdermal administration. Routes of administration for the EPAformulations that find use in the methods described herein include,e.g., oral (per os (P.O.)) administration, administration as asuppository, topical contact, transdermal delivery (e.g., via atransdermal patch), intravenous (“iv”) administration, intraperitoneal(“ip”) administration, or the implantation of a slow-release devicee.g., a mini-osmotic pump, a depot formulation, etc., to a subject.Administration can be by any route including parenteral and transmucosal(e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteraladministration includes, e.g., intravenous, intra-arterial,intraventricular. Other modes of delivery include, but are not limitedto, the use of liposomal formulations, intravenous infusion, transdermalpatches, etc.

The term “co-administering” or “concurrent administration”, when used,for example with respect to the EPA formulations described herein andanother active agent (e.g., pharmacological agents currentlyadministered to treat or ameliorate depression, hypertension, and/orelevated cholesterol levels, astaxanthin, vitamin E, phospholipids,coenzyme Q9 (CoQ9), coenzyme Q10 (CoQ10)), refers to administration ofEPA composition and the active agent such that both can simultaneouslyachieve a physiological effect. The two agents, however, need not beadministered together. In certain embodiments, administration of oneagent can precede administration of the other. Simultaneousphysiological effect need not necessarily require presence of bothagents in the circulation at the same time. However, in certainembodiments, co-administering typically results in both agents beingsimultaneously present in the body (e.g, in the plasma) at a significantfraction (e.g., 20% or greater, preferably 30% or 40% or greater, morepreferably 50% or 60% or greater, most preferably 70% or 80% or 90% orgreater) of their maximum serum concentration for any given dose.

The term “effective amount” or “pharmaceutically effective amount” referto the amount and/or dosage, and/or dosage regime of the EPAcompositions described herein necessary to bring about the desiredresult e.g., an amount sufficient to mitigate in a mammal one or moresymptoms associated with a disease condition mitigated by EPA (e.g.,depression), or an amount sufficient to lessen the severity or delay theprogression of a disease condition mitigated by EPA in a mammal (e.g.,therapeutically effective amounts), an amount sufficient to reduce therisk or delaying the onset, and/or reduce the ultimate severity of adisease condition mitigated by EPA in a mammal (e.g., prophylacticallyeffective amounts).

“Sub-therapeutic dose” refers to a dose of a pharmacologically activeagent(s), either as an administered dose of pharmacologically activeagent, or actual level of pharmacologically active agent in a subjectthat functionally is insufficient to elicit the intended pharmacologicaleffect in itself (e.g., to obtain analgesic and/or anti-inflammatoryeffects), or that quantitatively is less than the establishedtherapeutic dose for that particular pharmacological agent (e.g., aspublished in a reference consulted by a person of skill, for example,doses for a pharmacological agent published in the Physicians' DeskReference, 67th Ed., 2013, Thomson Healthcare or Brunton, et al.,Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12thedition, 2010, McGraw-Hill Professional). A “sub-therapeutic dose” canbe defined in relative terms (i.e., as a percentage amount (less than100%) of the amount of pharmacologically active agent conventionallyadministered). For example, a sub-therapeutic dose amount can be about1% to about 75% of the amount of pharmacologically active agentconventionally administered. In some embodiments, a sub-therapeutic dosecan be about 75%, 50%, 30%, 25%, 20%, 10% or less, than the amount ofpharmacologically active agent conventionally administered.

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the EPA compositions described herein to the subject. Causing to beadministered can involve diagnosis and/or determination of anappropriate therapeutic or prophylactic regimen, and/or prescribing theEPA compositions described herein for a subject. Such prescribing caninclude, for example, drafting a prescription form, annotating a medicalrecord, and the like.

The phrase “in conjunction with” when used in reference to the use ofthe EPA compositions described herein in conjunction with one or moreother active agent(s) so that there is at least some chronologicaloverlap in their physiological activity on the organism. When they arenot administered in conjunction with each other, there is nochronological overlap in physiological activity on the organism. Incertain preferred embodiments, the “other drug(s)” are not administeredat all (e.g., not co-administered) to the organism.

As used herein, the terms “treating” and “treatment” refer to delayingthe onset of, retarding or reversing the progress of, reducing theseverity of, or alleviating or preventing either the disease orcondition to which the term applies, or one or more symptoms of suchdisease or condition.

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease.

The term “psychiatric condition,” including the psychiatric conditionslisted herein, are as defined in the Diagnostic and Statistical Manualof Mental Disorders DSM-IV-TR Fourth Edition (Text Revision) by AmericanPsychiatric Association (June 2000).

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents recited in a method orcomposition (e.g., the EPA compositions described herein), and furthercan include other agents that, on their own do not substantial activityfor the recited indication or purpose. In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore additional agents that have pharmacological activity other than theEPA compositions and/or the listed components of the EPA compositionsdescribed herein.

The terms “subject,” “individual,” and “patient” interchangeably referto a mammal, preferably a human or a non-human primate, but alsodomesticated mammals (e.g., canine or feline), laboratory mammals (e.g.,mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g.,equine, bovine, porcine, ovine). In various embodiments, the subject canbe a human (e.g., adult male, adult female, adolescent male, adolescentfemale, male child, female child) under the care of a physician or otherhealthworker in a hospital, psychiatric care facility, as an outpatient,or other clinical context. In certain embodiments the subject may not beunder the care or prescription of a physician or other healthworker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a general overview of an illustrative process from aNannochloropsis paste to a standardized EPA and polar lipid mixture.

FIG. 2 illustrates the structure of phosphatidylcholine (PC), the mostcommon phospholipid in the Crude Algae Oil (CAO) and concentrated polarlipids (PoL).

FIGS. 3A-B illustrate the structures of A. MGDG(Monogalactosyldiacylglycerol), a single five carbon sugar ring, and B.DGDG (Digalactosyldiacylglycerol), a double five carbon sugar ring;glycolipids found in the herein described EPA formulations.

FIG. 4 illustrates an embodiment of extraction and refinement process toconvert Nannochloropsis paste to a standardized EPA and polar lipidmixture.

FIG. 5 illustrates an example of typical mass split of major mixtureconstituents during Nannochloropsis oil refining.

FIG. 6 illustrates an example of typical mass split of major mixtureconstituents during Nannochloropsis oil refining.

FIG. 7 illustrates a typical composition of S12 crude algae extract.

FIG. 8 illustrates a typical composition of S12 crude algae oil.

FIG. 9 illustrates a typical distribution of fatty acid by lipid classin S12 crude algae oil.

FIG. 10 illustrates a typical composition of S14 crude algae extract.

FIG. 11 illustrates a typical composition of S14 crude algae oil.

FIG. 12 illustrates a typical distribution of fatty acid by lipid classin S14 crude algae oil.

FIG. 13 illustrates free fatty acid (FFA) content of supercriticalcarbon dioxide fractionated partially hydrolyzed S14 neutral lipids.

FIG. 14 illustrates a distribution of characteristics of fatty acidmolecular weight chains and FFA as fractionated by supercritical carbondioxide.

FIG. 15 illustrates a typical composition of standardized EPAformulation that includes glycolipids, phospholipids, neutral lipids attriglycerides and diglycerides, and free fatty acid.

FIG. 16 illustrates a typical distribution of fatty acid acids by lipidclass in the standardized EPA formulation.

DETAILED DESCRIPTION

1. Introduction

Provided herein are compositions of nutritionally and pharmacologicallybeneficial mixtures comprising eicosapentanoic acid (EPA) Omega 3 fattyacids, polar lipids, and phytonutrients derived from Nannochloropsisoculata, an eustigmatophyte. These phytonutrients include Omega-7 fattyacid, chlorophyll, carotenoids, and coenzyme Q9 (CoQ9) and coenzyme Q10(CoQ10). N. Oculata is an eukaryotic algae that is unicellular withpolysaccharide cells walls and coccoid cells. Nannochloropsis contains ayellow-green chloroplast, which contains chlorophyll a, zeaxanthin, andbeta-carotene and specifically lacks chlorophyll b and c. The speciessynthesizes fatty acids in a number of different classes: neutral lipidscomprised of free fatty acid, triglycerides, and diglycerides and polarlipids comprised of phospholipids and glycolipids. Over two thirds ofthe fatty acids produced by Nannochloropis consist of eicosapentaenoicacid (EPA=C20:5ω3), palmitic acid (C16:0), palmitoleic acid(C16:1=C16:1ω7). The species produces only one other Omega-3,alpha-linolenic acid (ALA=C18:3ω3). Decosahexaenoic acid (DHA=C22:6ω3)is not produced by the species at all. Other Omega-3s fatty acids thatare notably absent are octadecatetraenoic acid or stearidonic acid(SDA=C18:4ω3), eicosatrienoic acid (ETE=C20:3ω3), eicosatetraenoic acid(ETA=C20:4ω3), heneicosapentaenoic acid or uncosapentaenoic acid(HPA=C21:5ω3), docapentaenoic acid (DPA=C22:5ω3).

Table 1 shows the relative fatty acid profile of fish oil ethyl esters(EPAX 6000 EE and EPAX 4020 EE), highly refined fish oil (MinamiNutrition Plus EPA), krill oil (NOW Neptune Krill Oil (NKO)), S12 andS14 variants of Nannochloropsis oculata. While the other sources includeat least seven of these eight Omega-3 fatty acids, Nannochloropsisoculata oil only contains two—EPA and ALA. In pure algal culture andunder the best growth conditions, the ratio of EPA to Total Omega3 isgreater than 99%. Under real-world conditions and in the presence ofthermal stress, the algae may produce additional ALA. The EPA to TotalOmega-3 ratio is greater than 90% and, more typically, greater than 93%,95%, 96%, 97%, or 98%. Other minor fatty acid components found in excessof 0.5% of the fatty acid profile are myristic (C14:0), Myristoleic(C14:1), Oleic (C18:1ω9), Oleic (C18:1ω7), linoleic (C18:2ω6), andarachidonic (C20:4ω6).

TABLE 1 Fatty Acid Profile From Different Omega-3 Sources C#:Dbl. FishOil Ethyl Esters Refined EPA Krill Oil Nannochloropsis Oculata FattyAcid Bond EPAX 6000 EE EPAX 4020 EE Plus EPA NOW NKO S12 S14 Caprylic 8:0 0.00 0.00 0.17 0.00 0.38 0.15 Capric 10:0 0.08 0.05 0.12 0.09 0.361.52 Lauric 12:0 0.00 0.00 0.00 0.18 0.70 0.41 Myristic 14:0 0.12 0.230.00 8.55 3.58 4.54 Myristoleic 14:1 0.00 0.00 0.00 0.36 4.26 0.09Pentadecanoic 15:0 0.00 0.00 0.00 0.31 0.22 0.36 Palmitic 16:0 0.49 2.510.00 18.69 13.12 23.48 Palmitoleic 16:1 0.43 0.97 0.00 5.74 16.51 24.32Hexadecadienoic 16:2 0.11 0.16 0.00 0.41 0.34 0.25 Hexadecatrienoic 16:30.16 0.21 0.00 0.00 0.00 0.00 Hexadecatetraenoic 16:4 0.10 0.27 0.000.49 0.00 0.00 Heptadecanoic 17:0 0.06 0.21 0.00 0.00 0.00 0.31 Stearic18:0 2.44 3.70 0.00 0.93 0.25 1.04 Oleic 18:1ω9 4.43 7.10 0.00 9.29 2.772.99 Oleic 18:1ω7 1.45 2.36 0.00 6.62 0.88 0.45 Linoleic 18:2ω6 0.370.83 0.00 1.92 4.04 2.40 Linoleic 18:2ω4 0.30 0.35 0.00 0.00 0.00 0.00Gamma-Linolenic 18:3ω6 0.00 0.19 0.00 0.00 0.25 0.48 Alpha-Linolenic(ALA) 18:3ω3 0.31 0.73 0.19 1.02 0.36 0.84 Octadecatetraenoic (SDA)18:4ω3 0.70 1.56 0.67 2.52 0.00 0.00 Arachidic 20:0 0.55 0.51 0.00 0.000.00 0.17 Eicosanoic 20:1ω11 0.36 0.28 0.00 0.09 0.00 0.00 Eicosanoic20:1ω9 2.07 2.08 0.00 0.77 0.00 0.00 Eicosanoic 20:1ω7 0.77 0.63 0.000.29 0.00 0.00 Eicosadienoic 20:2ω6 0.28 0.43 0.11 0.00 0.00 0.00Eicosatrienoic 20:3ω6 0.40 0.32 0.32 0.00 0.13 0.31 Eicosatrienoic (ETE)20:3ω3 0.16 0.21 0.07 0.00 0.00 0.00 Arachidonic 20:4ω6 2.09 1.91 3.590.64 3.97 4.79 Eicosatetraenoic (ETA) 20:4ω3 1.62 1.50 2.12 0.74 0.000.00 Eicosapentaenoic (EPA) 20:5ω3 41.89 33.30 88.89 18.09 35.62 19.73Uncosapentaenoic (HPA) 21:5ω3 2.22 1.68 1.11 0.45 0.00 0.00 Behenic 22:00.26 0.24 0.00 0.00 0.00 0.00 Erucic 22:1ω11 2.31 2.25 0.00 0.00 0.230.00 Erucic 22:1ω9 0.66 0.35 0.00 0.20 0.00 0.00 Docosatetraenoic 22:4ω60.00 0.00 0.00 0.27 0.00 0.00 Docosapentaenoic 22:5ω6 0.74 0.71 0.000.00 0.00 0.00 Docasapentaenoic (DPA) 22:5ω3 6.16 4.61 0.10 0.97 0.000.00 Docosahexaenoic (DMA) 22:6ω3 21.02 23.44 0.21 10.81 0.00 0.00Lignoceric 24:0 0.07 0.00 0.00 0.00 0.00 0.00 Nervonic 24:1 0.70 0.560.00 0.00 0.00 0.00 Other n/a 4.12 3.58 2.33 9.59 12.04 11.37 TotalFatty Acid (%) 100.0 100.0 100.0 100.0 100.0 100.0 Total Omega-3 (%)74.1 67.0 93.4 34.6 36.0 20.6 EPA in Total Omega-3 (%) 56.5 49.7 95.252.3 99.0 95.9 Total Omega-6 (%) 3.9 4.4 4.0 2.8 8.4 8.0

Provided herein are controlled formulations of total EPA, polar lipids,and phytonutrients. The formulations have the beneficial effects similarto that of krill oil, while including further advantages of glycolipids.Oil derived from krill contains solely phospholipids and no glycolipids.Animals, such as krill, synthesize phospholipids and do not synthesizeglycolipids. Plants synthesize phospholipids and glycolipids.Surprisingly, the present formulation is created from oil produced by asingle cell plant source that grows upon receiving sunlight and CO₂ toprovide a combination of high concentration of EPA fatty acids,phospholipids, and glycolipids. Furthermore, because of varyingenvironmental exposure, the chemical constituents with the algae canvary. To accommodate this variance, we have identified a means to createa controlled EPA concentration comprised of desirable amounts of EPA,phospholipids, glycolipids, and phytonutrients.

In varying embodiments, the controlled formulations are derived from twounique N. oculata strains, referred to herein as S12 and S14. Bothstrains originated in the University of Texas at Austin's UTEX TheCulture Collection of Algae (on the internet atweb.biosci.utexas.edu/utex/). S12 is adapted for lower temperatureenvironmental conditions while S14 is more tolerant of highertemperature conditions. The N. oculata can be grown in outdoor culturein a raceway cultivation system, known in the art. The biomass is amarine algae growing in a saltwater solution at dilute concentrationsbetween 1 and 20 g/L. At harvest, the algae is concentrated through anumber of techniques known to those in the art, including combinationsof cross-flow filtration, flocculation, settling, dissolved airfloatation, and centrifugation. Following harvest, the resulting solidsconcentrate is in the range of 100 g/L (10 wt %) to 300 g/L (30 wt %)solids. When centrifugation is employed, the concentration is moretypically in range of 18 to 25 wt % solids. We refer to this materialform as algae paste. When the algal biomass is predominantlyNannochloropsis, it is called Nanno Paste.

The creation of mixture of EPA and polar lipids requires the executionof a series of extraction and refinement steps. This includes biomassextraction, removal of non-lipid and water-soluble components (MONLRefinement), separation of neutral and polar lipid constituents,concentration of the EPA in the neutral lipid fraction, and blending toachieve a standard EPA concentration in the final product. This genericprocess is shown in FIG. 1. The EPA fatty acid exists in thestandardized product conjugated with glycolipids, phospholipids,triglycerides, and as a non-conjugated free fatty acid (FFA). Biomassextraction involves the isolation of a liquid solution comprisedpredominantly of lipid with minor components of protein, carbohydrate,mineral, and fiber of the biomass. This process creates a lipid mixturecalled crude algae extract (CAE), a complex mixture of lipids,phytonutrients, carbohydrates, water-soluble proteins, and water. CAEcontains a large fraction of constituents other than lipids andphytonutrients—Matter Organic-Not Lipid (MONL). The next process removesbetween half and greater than 90% of the MONL component, creating CrudeAlgae Oil (CAO). This intermediate material is one of the threecomponents of the final blend, and contains neutral lipids, polarlipids, and phytonutrients.

We have found that the Nannochloropsis contains between 5 and 50 mg/kg(ppm) CoQ9 and between 20 and 100 ppm CoQ10. In S12, we have measuredCoQ9 at 8.5 and 25 ppm. In S14, we have measured CoQ9 at 19 ppm. ForS12, we have measured CoQ10 at 31 ppm and 35 ppm. For S14, CoQ10 wasmeasured at 67 ppm. CoQ9 and CoQ10 represent phytonutrients or minormicronutrients present in the Nannochloropsis extract.

The next process step separates the neutral and polar lipids. The polarlipid (PoL) concentrate contains virtually all the phospholipid and theglycolipid constituents present in the CAO. The neutral lipid fractionis processed in Neutral Lipid Homogenization. This process converts thefatty acids to a single molecular form that is not conjugated with aglycerol backbone. The homogenized stream now consists of a uniformlipid type with the most common forms being salts and FFAs. The lipidfractionation step concentrates the higher molecular weight and greaterdouble bond of the EPA fatty acids from the other fatty acids in thedistribution. One form for blending is FFAs. Thus, if salts are employedin the fractionation, the salts are acidified to form FFAs. TheConcentrated EPA FFA (EPA-FFA) is the third components of the blend. Thecontrolled formulation is a blend of CAO, PoL Concentrate, and EPA-FFAConcentrate. The individual constituents are characterized for theirfatty acid profile and polar lipid profile. The mass ratio of eachconstituent is adjusted to meet a target PoL and EPA content.

The PoL concentrate is comprised in greater than 30% total polar lipids.The amount of glycolipid is between 1 and 3 times the phospholipid. Thephospholipid consistent, typically, of phosphorous and other organicmoiety conjugated with the glycerol backbone in the SN3 position and oneor two fatty acids in the SN1 and SN2 (middle) position on the glycerolbackbone. Phosphatidylcholine (PC), the most common phospholipid in theCAO and concentrated PoL, is shown in FIG. 2. Note that the fatty acidin SN1 position is the R¹ group and the carboxylic acid (COO) group.Similarly, the fatty acid in the SN2 position is the R² group and theCOO group. When EPA is associated with the phospholipid class, it is ineither the SN1 or SN2 position. The glycolipids are shown in FIG. 3consist of MGDG (Monogalactosyldiacylglycerol), a single five carbonsugar ring, or DGDG (Digalactosyldiacylglycerol), a double five carbonsugar ring. In a similar fashion, the glycolipid associated fatty acidsare in SN1 position as shown by the OCOR¹ moiety and SN2 position asshown by the —OCOR² moiety. The fatty acids conjugated with glycolipidsare in these two positions. The EPA fatty acid associated with theConcentrated PoL and CAO is measured. Concentrate EPA-FFA is blendedwith the other two components to achieve a target EPA amount of at least25 wt %. EPA concentration in the PoL and the CAO is less than thisvalue. A typical EPA value in the COA and concentrated PoL is 14 wt %and 11 wt %, respectively. The Concentrated EPA-FFA is greater than 40%concentration and, thus, can be blended with lower EPA concentrations toachieve the 25 wt % concentration. The EPA content of the mixture isdetermined by rule of mixtures, the sum of products of the mass fractionof each blend component and EPA concentration.

The standardized formulation is advantageous due to increasedbioavailability as relates to metabolic functions resulting in lipidabsorbance into the body (on the internet atvivo.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html).As fats move through the lumen of the small intestine, they must passthrough the cell membrane of the enterocytes, the columnar epithelialcells lining the small intestine and colon, to be absorbed into thebody. The triglyceride (TG) and, to some extent, the diglyceride (DG)neutral fats are hydrophobic and insoluble in water. When a mixture ofTG and DGs is exposed to water, these molecules are attracted to eachother and repelled by the water, forming large micelles that disperse inthe water. These large micelles are incapable of diffusing across theplasma membrane due to size exclusion. Polar lipids, such asphospholipids (PL) and glycolipids (GL), have a glycerol backbone thatlinks the hydrophobic fatty acid moiety with the hydrophilic phosphorusor carbohydrate moiety. These polar lipids are amphipathic. Along withthe bile acids in the intestine, PL and GL aid in emulsifying TG and DGneutral fats. The net effect is to break the TG/DG micelles intomultiple smaller micelles, thus preserving mass while increasing themicelle surface area. The presence of TG, PL, and GL is critical, astheir presence is the trigger for the release of pancreatic lipase, awater-soluble enzyme. Pancreatic lipase acts on the SN1 and SN3 positionof the triglyceride and hydrolyzes the fatty acids in these positions,creating free fatty acids (FFA) and 1-monoglyceride (1-MG), a glycerolbackbone with a fatty acid remaining in the SN2 position. FFA areamphipathic like PLs and GLs. The lipases also act on the polar lipidsto cleave the fatty acids in SN1 position, forming a 1 lysopholipids(1-PL) such as 1-lysophosphatidylcholine (1-LPC) and1-lysophosphatidylethanolamine (1-LPE), and 1-lysoPhosphatidylinositol(1-LPI). FFA, 1-MG, and 1-PL can then enter into the enterocytes viadiffusion or via a fatty acid transporter protein in the enterocytemembrane. Because the EPA-FFA is a smaller molecule of roughly one thirdthe molecular weight of the TG, PL, or GL and amphipathic, EPA-FFA canbe absorbed without enzymatic and/or bile action. EPA is furtherabsorbed via the TG, DG, PL, and GL routes. The formulation containsphytonutrients including chlorophyll, sterols, and carotenoids that arein admixture with the TG and DG, preventing oxidative degradation priorto absorbance. Sterols are noted for inhibiting the uptake ofcholesterol in the intestinal tract.

2. EPA Formulations

Generally, the EPA formulations comprise in the range of about 15 wt. %to about 90 wt. % eicosapentaenoic acid (EPA), e.g., from about 20 wt. %to about 75 wt. % EPA, e.g., from about 20 wt. % to about 50 wt. % EPA,in its various chemical forms (e.g., as FFA, diglyceride, triglyceride,phospholipid, glycolipid); in the range of about 10 wt. % to about 70wt. % polar lipids (glycolipids and phospholipids), e.g., from about 30wt. % to about 35 wt. % polar lipids, and do not compriseDocosahexaenoic acid (DHA). The present EPA compositions are formulatedfor human consumption and for improved bioavailability of EPA byincreasing the proportion of EPA in its most bioavailable forms (e.g.,as a free fatty acid, as a phospholipid conjugate, and/or as aglycolipid conjugate), and reducing or eliminating EPA in its lessbioavailable forms (e.g., as a diglyceride conjugate and/or as atriglyceride conjugate). The compositions further do not compriseesterified fatty acids, including esterified EPA.

In varying embodiments, the EPA is in one or more forms (e.g., 2, 3, 4or all forms) selected from the group consisting of a phospholipidconjugate, a glycolipid conjugate, a triglyceride conjugate, adiglyceride conjugate and/or free fatty acid. In varying embodiments,EPA in the form of a triglyceride conjugate and/or a diglycerideconjugate is reduced to less than about 0.2 wt. % or to non-detectablelevels, or completely eliminated.

In varying embodiments, the EPA to total omega-3 fatty acids ratio isgreater than 90%, e.g., greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more. In some embodiments, the composition comprises about25 wt. % to about 50 wt. % EPA, e.g., about 25 wt. %, 30 wt. %, 35 wt.%, 40 wt. %, 45 wt. % or 50 wt. % EPA.

With respect to the distribution of fatty acids by lipid class in thecompositions formulated for human consumption, in varying embodiments,the compositions comprise from about 10 wt. % to about 15 wt. %phospholipids (e.g., about 10 wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14wt. %, 15 wt. % phospholipids), from about 15 wt. % to about 25 wt. %glycolipids (e.g., about 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt.%, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24 wt. %, 25 wt. %glycolipids), from about 0 wt. % to about 10 wt. % di- andtri-glycerides (e.g., less than about 10 wt. %, 9 wt. %, 8 wt. %, 7 wt.%, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.5 wt. % di-and tri-glycerides), and from about 30 wt. % to about 45 wt. % freefatty acids (e.g., about 30 wt. %, 31 wt. %, 32 wt. %, 33 wt. %, 34 wt.%, 35 wt. %, 36 wt. %, 37 wt. %, 38 wt. %, 39 wt. %, 40 wt. %, 41 wt. %,42 wt. %, 43 wt. %, 44 wt. %, 45 wt. % free fatty acids). In varyingembodiments, the compositions comprise about 30-35 wt. % (e.g., about ⅓)polar lipids (i.e., phospholipids and glycolipids combined). In varyingembodiments, the compositions do not have detectable levels of, havebeen isolated from and/or are free of di- and tri-glycerides. In varyingembodiments, the compositions comprise less than about 0.2 wt. % of di-and tri-glycerides. Accordingly, in some embodiments, the compositionscomprise from about 10 wt. % to about 15 wt. % phospholipids (e.g.,about 10 wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %phospholipids), from about 15 wt. % to about 25 wt. % glycolipids (e.g.,about 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt.%, 22 wt. %, 23 wt. %, 24 wt. %, 25 wt. % glycolipids), less than about0.2 wt. % di- and tri-glycerides, and from about 30 wt. % to about 45wt. % free fatty acids (e.g., about 30 wt. %, 31 wt. %, 32 wt. %, 33 wt.%, 34 wt. %, 35 wt. %, 36 wt. %, 37 wt. %, 38 wt. %, 39 wt. %, 40 wt. %,41 wt. %, 42 wt. %, 43 wt. %, 44 wt. %, 45 wt. % free fatty acids).

With respect to the distribution of EPA by lipid class in thecompositions formulated for human consumption, in varying embodiments,the compositions comprise from about 3 wt. % to about 30 wt. %, e.g.,from about 5 wt. % to about 20 wt. %, as phospholipid conjugate (e.g.,about 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %, 16 wt. %, 17wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30 wt. % asphospholipid conjugate), from about 8 wt. % to about 50 wt. %, e.g.,from about 10 wt. % to about 25 wt. %, as glycolipid conjugate (e.g.,about 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %, 16 wt. %, 17wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30 wt. %, 31wt. %, 32 wt. %, 33 wt. %, 34 wt. %, 35 wt. %, 36 wt. %, 37 wt. %, 38wt. %, 39 wt. %, 40 wt. %, 41 wt. %, 42 wt. %, 43 wt. %, 44 wt. %, 45wt. %, 46 wt. %, 47 wt. %, 48 wt. %, 49 wt. %, 50 wt. % as glycolipidconjugate), from about 0 wt. % to about 10 wt. % di- and tri-glycerideconjugates (e.g., less than about 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, or 0.5 wt. % as di-and tri-glyceride conjugates), and from about 40 wt. % to about 85 wt.%, e.g., from about 50% wt. % to about 80 wt. %, as free fatty acids(e.g., at least about 40 wt. %, 41 wt. %, 42 wt. %, 43 wt. %, 44 wt. %,45 wt. %, 46 wt. %, 47 wt. %, 48 wt. %, 49 wt. %, 50 wt. %, 51 wt. %, 52wt. %, 53 wt. %, 54 wt. %, 55 wt. %, 56 wt. %, 57 wt. %, 58 wt. %, 59wt. %, 60 wt. %, 61 wt. %, 62 wt. %, 63 wt. %, 64 wt. %, 65 wt. %, 66wt. %, 67 wt. %, 68 wt. %, 69 wt. %, 70 wt. %, 71 wt. %, 72 wt. %, 73wt. %, 74 wt. %, 75 wt. %, 76 wt. %, 77 wt. %, 78 wt. %, 79 wt. %, 80wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, or 85 wt. % as free fattyacids). In varying embodiments, the compositions comprise about from 10wt. % to about 50 wt. %, e.g., from about 15 wt. % to about 30 wt. %polar lipids (i.e., phospholipids and glycolipids combined). In varyingembodiments, the compositions do not have detectable levels of, havebeen isolated from and/or are free of di- and tri-glycerides. In varyingembodiments, the compositions comprise less than about 0.2 wt. % of di-and tri-glycerides. Accordingly, in some embodiments, the compositionscomprise EPA distributed by lipid class as from about 3 wt. % to about30 wt. %, e.g., from about 5 wt. % to about 20 wt. %, phospholipidconjugate (e.g., about 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8wt. %, 9 wt. %, 10 wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt.%, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %,23 wt. %, 24 wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30wt. % phospholipid conjugate), from about 8 wt. % to about 50 wt. %,e.g., from about 10 wt. % to about 25 wt. %, glycolipid conjugate (e.g.,about 3 wt. %, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %, 16 wt. %, 17wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30 wt. %, 31wt. %, 32 wt. %, 33 wt. %, 34 wt. %, 35 wt. %, 36 wt. %, 37 wt. %, 38wt. %, 39 wt. %, 40 wt. %, 41 wt. %, 42 wt. %, 43 wt. %, 44 wt. %, 45wt. %, 46 wt. %, 47 wt. %, 48 wt. %, 49 wt. %, 50 wt. % glycolipidconjugate), less than about 0.2 wt. % di- and tri-glyceride conjugates,and from about 40 wt. % to about 85 wt. %, e.g., from about 50% wt. % toabout 80 wt. %, free fatty acids (e.g., about 40 wt. %, 41 wt. %, 42 wt.%, 43 wt. %, 44 wt. %, 45 wt. %, 46 wt. %, 47 wt. %, 48 wt. %, 49 wt. %,50 wt. %, 51 wt. %, 52 wt. %, 53 wt. %, 54 wt. %, 55 wt. %, 56 wt. %, 57wt. %, 58 wt. %, 59 wt. %, 60 wt. %, 61 wt. %, 62 wt. %, 63 wt. %, 64wt. %, 65 wt. %, 66 wt. %, 67 wt. %, 68 wt. %, 69 wt. %, 70 wt. %, 71wt. %, 72 wt. %, 73 wt. %, 74 wt. %, 75 wt. %, 76 wt. %, 77 wt. %, 78wt. %, 79 wt. %, 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85wt. % free fatty acids).

In varying embodiments, the glycolipids comprise one or more ofdigalactosyldiacylglycerol and monogalactosyldiacylglycerol. In someembodiments, the phospholipids comprise one or more ofphosphatidylcholine, lyso-phosphatidylcholine, phosphatidylinositol,phosphatidylethanolamine and phosphatidylglycerol. In some embodiments,the phospholipids comprise one or more of phosphatidylcholine andphosphatidylglycerol.

In some embodiments, the EPA compositions comprise:

i) 0 to 5 wt. % C:18 fatty acids, e.g., 0.2 to 3 wt. % C:18 fatty acids;

ii) 0 to 20 wt. % C:16 fatty acids, e.g., 2 to 20 wt. % C:16 fattyacids;

iii) 0 to 5 wt. % C:14 fatty acids, e.g., 0.2 to 5 wt. % C:14 fattyacids;

iv) 0 to 0.2 wt. % C:12 fatty acids; and/or

v) 0 to 0.1 wt. % C:10 fatty acids.

In varying embodiments, the composition comprises:

Component wt. % Fatty Acids Capric (10:0) 0.1-0.2 Lauric (12:0) 0.1-0.2Myristic (14:0) 1.0 Palmitic (16:0) 5.3-6.5 Palmitoleic (16:1) 7.0-8.0Hexadecadienoic (16:2) 0.2-0.3 Stearic (18:0) 0.2-0.3 Oleic (18:1ω9) 2.0Oleic (18:1ω7) 0.3-0.4 Linoleic (18:2ω6) 1.2-2.0 Alpha-Linolenic (ALA)(18:3ω3) 0.2 Arachidonic (20:4ω6) 1.0-8.0 Eicosapentaenoic (EPA)(20:5ω3) 19-30 Total Fatty Acid 40-55 Total Omega-3 20-30EPA/Omega-3 >93    Total Omega-6  2-10 Phospholipids Phosphatidylcholine4.7-7.4 Lyso-Phosphatidylcholine 0.3-0.4 Phosphatidylinositol 0.8-1.3Phosphatidylethanolamine 0.5-0.8 Phosphatidylglycerol 1.8-2.8Glycolipids Digalactosyldiacylglycerol 10-17Monogalactosyldiacylglycerol 3-5 Phytonutrients Phytosterols 1.5Chlorophyll 4-5 Total Phospholipids (PL) (wt %)  9-14 Total Glycolipids(GL) (wt %) 13-21 Total PoL (PL + GL) (wt %) 22-35

In varying embodiments, the composition comprises chlorophyll a. Invarying embodiments, the composition comprises less than about 10.0 wt.% arachidonic acid, e.g., less than about 9.5 wt. %, 9.0 wt. %, 8.5 wt.%, 8.0 wt. %, 7.5 wt. %, 7.0 wt. %, 6.5 wt. %, 6.0 wt. %, 5.5 wt. %, 5.0wt. %, 4.5 wt. %, 4.0 wt. %, 3.5 wt. %, 3.0 wt. %, 2.5 wt. %, 2.0 wt. %,1.5 wt. % or 1.0% arachidonic acid, or does not comprise arachidonicacid (i.e., 0 wt. %).

In varying embodiments, the composition does not comprise or issubstantially free of intact cells, cellular components,polynucleotides, and polypeptides. In varying embodiments, thecomposition does not comprise fatty acids selected from the groupconsisting of octadecatetraenoic acid or stearidonic acid (SDA=C18:4ω3),eicosatrienoic acid (ETE=C20:3ω3), eicosatetraenoic acid (ETA=C20:4ω3),heneicosapentaenoic acid or uncosapentaenoic acid (HPA=C21:5ω3), anddocapentaenoic acid (DPA=C22:5ω3). In varying embodiments, thecomposition does not comprise carotenoids selected from the groupconsisting of astaxanthin, cis-lutein, trans-lutein, cis-zeaxanthin,trans-alpha-cryptoxanthin, trans-alpha-carotene, cis-alpha-carotene,cis-lycopene, and trans-lycopene. In varying embodiments, thecomposition does not comprise chlorophyll c. In varying embodiments, thecomposition does not comprise one or more phospholipids selected fromthe group consisting of N-acyl-phosphatidylethanolamine,lyso-phosphatidylcholine, phosphatidylinositol andphosphatidylethanolamine. In some embodiments, the composition does notcomprise sphingolipids.

The compositions may further comprise a pharmaceutically acceptablecarrier and/or one or more pharmaceutically acceptable excipients.Generally the compositions are not biphasic (e.g., are monophasic), andcomprise less than about 10% water, e.g., less than about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1% water, or no water.

Also contemplated are capsules, tablets, solutions, syrups, andsuspensions suitable for human consumption comprising the EPAcomposition described above and herein. In varying embodiments, thecapsule is a gelatin capsule or a soft capsule, including soft capsulesfrom non-animal, vegetarian sources. Further contemplated are foods,beverages, energy bars, and nutritional supplements comprising the EPAcompositions described herein.

3. Methods of Producing Highly Bioavailable EPA Formulations

The methods provide for the energy and cost efficient production of EPAformulations derived from microalgal oils (e.g., from Nannochloropsis)that maximize the amounts of EPA in its most bioavailable forms (e.g.,as a free fatty acid or as a glycolipid or phospholipid conjugate).Schematics illustrating steps for preparing the EPA formulationsdescribed herein are provided in FIGS. 1 and 4.

In varying embodiments, the methods comprise the steps of:

a) providing an algal paste;

b) extracting lipids from the algal paste with an organic solvent,thereby substantially isolating a crude algae extract (CAE) comprisingneutral lipids and polar lipids from the water-soluble components of thepaste;

c) substantially removing the water-soluble components, thereby yieldinga crude algae oil (CAO);

d) hydrolyzing a first portion of the CAO, thereby releasing free fattyacids in the portion of CAO;

e) fractionating the released free fatty acids according to chainlength, thereby isolating C20 free fatty acids and yielding aconcentrated EPA free fatty acid fraction; and

f) combining the concentrated EPA free fatty acid fraction produced instep e) and a second portion of the CAO produced in step c).

In varying embodiments, the methods comprise the steps of:

a) providing an algal paste;

b) extracting lipids from the algal paste with an organic solvent,thereby substantially isolating a crude algae extract (CAE) comprisingneutral lipids and polar lipids from the water-soluble components of thepaste;

c) substantially removing the water-soluble components, thereby yieldinga crude algae oil (CAO);

d) contacting the CAO with supercritical CO2, wherein the supercriticalCO2 selectively extracts the neutral lipids, thereby splitting the CAOinto a neutral lipid fraction comprising free fatty acids and a polarlipid fraction comprising glycolipids and phospholipids;

e) isolating C20 free fatty acids from the neutral lipid fraction,thereby yielding a concentrated EPA free fatty acid fraction; and

f) combining the concentrated EPA free fatty acid fraction produced instep e) and the polar lipid fraction produced in step d).

Generally, the methods do not comprise the steps of disrupting algalcells, subjecting algal cells to mechanical cracking, thermalpretreatment, alkaline treatment and/or acid treatment. In addition, thesteps specifically avoid esterifying the fatty acids, including withoutlimitation the conversion to methyl esters or ethyl esters.

The methods can be used to produce highly bioavailable EPA compositionsfrom any biomass source of EPA. Preferably, the biomass source producesoils having EPA at a concentration in the range of about 30 wt. % toabout 70 wt. %. For example, EPA is naturally produced in a variety ofnon-oleaginous and oleaginous microorganisms, including theheterotrophic diatoms Cyclotella sp. and Nitzschia sp. (U.S. Pat. No.5,244,921), Pseudomonas, Alteromonas and Shewanella species (U.S. Pat.No. 5,246,841), filamentous fungi of the genus Pythium (U.S. Pat. No.5,246,842), Mortierella elongata, M. exigua, and M. hygrophila (U.S.Pat. No. 5,401,646), and eustigmatophycean alga of the genusNannochloropsis (Krienitz, L. and M. Wirth, Limnologica, 36:204-210(2006)). Moreover, several types of yeast have been recombinantlyengineered to produce EPA. See for example, work in the non-oleaginousyeast Saccharomyces cerevisiae (U.S. Pat. No. 7,736,884) and theoleaginous yeast, Yarrowia lipolytica (U.S. Pat. Nos. 7,238,482;7,932,077; U.S. Pat. Appl. Pub. No. 2009-0093543-A1; U.S. Pat. Appl.Pub. No. 2010-0317072-A1). In varying embodiments, the biomass sourcecan be fish or krill oil.

a. Providing an Algal Paste

In one step, algal cells are harvested and concentrated to form a paste.In various embodiments, a paste of the algal cell source material isprovided.

In varying embodiments, the EPA formulations are derived from algalcells and do not contain any docosahexaenoic acid (DHA). In varyingembodiments, the source biomass for the EPA compositions is from amicroalgae of the genus Nannochloropsis. In varying embodiments thesource biomass is from a Nannochloropsis selected from the groupconsisting of Nannochloropsis gaditana, Nannochloropsis granulate,Nannochloropsis limnetica, Nannochloropsis maritime, Nannochloropsisoceanica, Nannochloropsis oculata, Nannochloropsis salina, andNannochloropsis sp. (e.g., 10S010, AN1/12-10, AN1/12-5, AN1/12-7,AN2/29-2, AN2/29-6, AS4-1, BR2, C95, CCAP211/46, CCAP211/78, CCMP1779,CCNM 1032, CCNM 1034, CSIRO P74, HSY-2011, JL11-8, JL2/4-1, KMMCCEUS-02, KMMCC EUS-05, KMMCC EUS-06, KMMCC EUS-08, KMMCC EUS-09, KMMCCEUS-11, KMMCC EUS-12, CCMP2195, KMMCC EUS-13, KMMCC EUS-14, KMMCCEUS-15, KMMCC EUS-16, KMMCC EUS-17, KMMCC EUS-18, KMMCC EUS-19, CCMP533,KMMCC EUS-20, KMMCC EUS-21, LL-2012, MA-2012, UTEX2164, MBTD-CMFRI-5006,MBTD-CMFRI-5007, MBTD-CMFRI-S012, MBTD-CMFRI-5076, MBTD-CMFRI-5077,MBTD-CMFRI-5078, CCMP525, MDL11-16, MDL3-4, NANNO-IOLR, RCC438,CCAP849/7, RCC504, SC-2012, strain IOLR, Tow 2/24 P-1w, UTEX2379, W2J3B,YJH-2012, W0980). In varying embodiments, the source biomass is from aNannochloropsis selected from the group consisting of Nannochloropsisoceanica, and Nannochloropsis oculata.

Microalgae, e.g., Nannochloropsis grows in relatively dilute culturethat is typically in the range of 0.1 to 1.0 g/L of biomass and, moretypically, in the range of 0.3 to 0.7 g/L. For a 0.5 g/L cultureconcentration, this implies that there is 0.5 g of dry weight equivalentbiomass for every 1000 g of culture, a dilute concentration. Algae arefurther processed in a more concentrated state, typically in the rangeof 2 to 300 g/L. The microalgae are harvested from culture andconcentrated into a paste using any method known in the art. In varyingembodiments, dewatering, sedimentation, filtration, cross-flowfiltration and/or centrifugation techniques that are known in the artcan be employed. As appropriate, air sparging and flocculationtechniques can be employed to facilitate concentration and harvesting ofNannochloropsis cells.

Methods for harvesting and concentrating Nannochloropsis cells are knownin the art and find use. See, e.g., U.S. Patent Publ. Nos. 2013/0046105;2012/0282651; 2012/0225472; 2012/0108793; and 2011/0081706 and Sirin, etal., Bioresour Technol. (2013) January 22; 132C:293-304; Farid, et al.,Bioresour Technol. 2013 January 23. doi:pii: S0960-8524(13)00081-3; andWan, et al., Bioresour Technol. 2012 Oct. 16. doi:pii:S0960-8524(12)01506-4. The foregoing references are hereby incorporatedherein in their entirety for all purposes.

In one embodiment, Nannochloropsis cells are harvested and concentratedby raising the pH of the culture fluid, e.g., to about pH 10.0, exposingthe cells to a flocculant and/or coagulant, thereby concentrating thecellular biomass from about 0.5 g/L to about 10-20 g/L biomass. Thecoagulated/flocculated cells are allowed to settle, e.g., in a settlingtank, the aqueous supernatant above the settled cellular biomass isdecanted and the remaining fluid containing the settled cellular biomassis further dewatered by centrifugation, thereby forming an algal paste.

b. Extracting Lipids from Algal Paste with an Organic Solvent Lipids

Lipids can be extracted from the algal paste in either the wet or drystate using any method known in the art. In the wet state, the moisturecontent is between 400 and 1000% (w/w) dried biomass (25 to 10 wt %solids). In the dry state, the moisture content is less than 15% (w/w)of the dried biomass. Lipids can be extracted from the algal biomassusing an organic solvent. In varying embodiments, about 1× to about 20×,e.g., about 2× to about 7×, the mass of organic solvent is mixed withthe biomass to form a biomass, solvent, and extract slurry. The algalpaste can be exposed to, contacted with and/or submerged in the solventwithout pretreatment steps.

Surprisingly, we have found that biomass extraction to obtain the crudealgae extract requires no mechanical cracking (such as a bead mill),thermal pretreatment, or cellular wall digestion, e.g., via acid orbase. In some embodiments, the lipids are extracted from wet state algalpaste. For wet extraction of biomass, a pure solvent or solvent mixturethat is at least partially miscible with water is used. We haveunexpectedly discovered that the extract from wet algal paste leads tobetween 1.5 and 3.5 times more fatty acid recovery from the biomassversus the extraction of the same biomass after drying. Even with noparticular effort to disrupt the cell membrane via mechanical, thermal,or pH disruption (e.g., alkaline or acid treatment), the wet paste has ahigher extraction yield than the same biomass after drying.

Solvents useful for extraction of lipids from the algal paste include abroad selection of solvents types, including ethers, ketones, andalcohols. Solvents and solvent mixtures of use have the ability toextract hydrophobic, non-polar lipid components such as triglyceridesand hydrophilic, polar lipid components such as phospholipids andglycolipids from the algal paste. Illustrative solvent systems includeethanol, isopropyl alcohol, acetone and ethanol, dimethyl ether,dimethyl ether and ethanol. In varying embodiments, the solvent systemis either an ether and alcohol mixture or a ketone and alcohol mixture.Illustrative solvent combinations of use include absolute ethanol, 190proof (95 v/v %) ethanol (EtOH), denatured 190 proof ethanol, specialdenatured alcohols (SDA), acetone and ethanol, isopropyl alcohol,acetone and methanol, methyl ethyl ketone (MEK) and methanol, MEK andethanol, dimethyl ether, dimethyl ether and methanol, dimethyl ether andethanol. In varying embodiments, lipids are extracted from the algalpaste using a solvent mixture that is 50 wt. % (v/v) acetone and 50 wt.% (v/v) 190 proof ethanol (EtOH). Other solvent mixtures of use includepure dimethyl ether (DME), DME mixed with methanol, or solely 190 proofEtOH. EtOH may be non-denatured or one of the Special Denatured Alcohol(SDA) grades (1-1, 1-2, 2B-2, 2B-3, 3A, 3C, 23A, 23H, 29, 30, 35A) proofdenatured ethanol, where the major composition of the SDAs is given inTable 5.

Extraction can be performed by any method known in the art, includingbatchwise and continuous flow methods (e.g., countercurrent columns,crossflow filtration). Solvent percolation through the biomass paste canbe facilitated by mixing paste with a filtration aid (e.g., diatomaceousearth) or by vigorous mixing with solvent coupled with crossflowfiltration. The extracted lipid-rich solution can be separated from thebiomass using any method known in the art, e.g., filtration orcentrifugation, where filtration or, in some embodiments, cross-flowfiltration is employed for removing solid from the solution. For nearlycomplete lipid extraction, multiple stages of extraction are performed,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stages, as appropriate.Extraction of lipid components from the algal paste using an organicsolvent creates a crude algae extract (CAE). Crude algae extract (CAE)can contain between 10 and 80% of constituents that are not lipids. CAEgenerally comprises neutral lipids, polar lipids, chlorophyll, sterols,carotenoids, manitol, and glycerol.

c. Removing Water-soluble Components from the CAE to Produce Crude AlgalOil (CAO)

The CAE contains a significant proportion of non-lipid organic material(“matter-organic non-lipid” or “MONL”). MONL is material not otherwiseaccounted for in the total fatty acids (TFA), phospholipids,glycolipids, and phytonutrients. MONL contains water-soluble components,e.g., water-soluble carbohydrates and proteins. Varying embodiments ofthe methods perform the step of substantially removing water-solublecomponents from the CAE to produce a crude algal oil (CAO).Water-soluble components can be substantially removed from CAE, therebyyielding CAO, through any method known in the art, e.g., through organicsolvent and water partitioning. This approach separates the highly polarwater-soluble constituents from the non-polar (e.g. neutral lipids) andmixed polarity (e.g., PL and GL) constituents. In varying embodiments,CAE recovered from solvent extraction can be solubilized in anothersolvent and then added to a liquid-liquid partitioning system tosubstantially remove water-soluble components.

In varying embodiments, MONL refinement may include a partition of thewater-soluble components comprising excess water and organic solvent toCAE, bringing the water, organic solvent, and CAE into intimate contactwith a high shear mixer, and separation of the water and organic phasevia either settling or centrifugation. After agitation to assumeintimate contact between the feed, the water and organic phase areseparated by either a settling tank or centrifugation (i.e. throughenhanced gravity). The material splits into an upper organic layer andlower aqueous layer. The neutral and polar lipids, sterols, andcholesterol have a much higher distribution coefficient for the organiclayer and predominantly remain in the organic layer. Water-solubleconstituents, including carbohydrates (especially mannitol),water-soluble proteins, and glycerol predominantly, go into solutionwithin the aqueous layer. The organic phase is the CAO which contains alipid rich mixture of polar lipids (PoL) and neutral lipids (NL).

Alternatively, CAE can be extracted in series with a solvent moresuitable for neutral lipids followed by further extraction by a solventsuitable for polar lipids. Illustrative solvents suitable for extractionof neutral lipids include hexane, chloroform, cyclohexane, methylenechloride, carbon dioxide or combinations thereof. Illustrative solventssuitable for extraction of polar lipids include acetone, methanol,ethanol or combination thereof. A further useful solvent combinationincludes heptane (Hep), ethyl acetate (EtAc), methanol (MeOH), and water(H₂O), e.g., in a volume ratio of 1:1:1:1.

In varying embodiments liquid-liquid partitioning can employ alternateenvironmentally friendly organic solvents. Illustrative environmentallyfriendly solvents include without limitation water, acetone, ethanol,2-propanol, 1-propanol, ethyl acetate, isopropyl acetate, methanol,methyl ethyl ketone (MEK), 1-butanol, and t-butanol. Other solvents ofuse for liquid-liquid partitioning include liquid of cyclohexane,heptane, toluene, methylcylcohexane, methyl t-butyl ether, isooctane,acetonitrile, 2-methyltetrahydrofuran, tetrahydrofuran (THF), xylenes,dimethyl sulfoxide (DMSO), acetic acid, and ethylene glycol.

In varying embodiments, the solution partitions and the NL-rich upperphase is collected. A NL-rich extract is recovered by evaporating thesolvent. The bottom phase, now rich in both PoL and MONL, is extractedwith a PoL suitable solvent system. A PoL-rich extract is recovered byseparating the hydrophobic layer from the hydrophilic layer andevaporating off the solvents. CAO results when the NL-rich and PoL-richextracts are combined. Substantial removal of water-soluble componentsto effect the conversion from CAE to CAO results in between 30% and 50%reduction in mass. CAO is one of the constituents of the EPAstandardized EPA/Polar Lipid blend. In varying embodiments, a portion ofthe CAE is included in the standardized EPA blend.

In varying embodiments, at least about 90% of the water-solublecomponents, e.g. at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more of the water-soluble components are removed from orseparated from the crude algae extract to yield the crude algae oil.

d. Optionally Extracting Neutral Lipids from Crude Algal Oil withSupercritical CO₂

In varying embodiments, the CAO can be converted into a neutral lipid(NL)-rich stream that contains no polar lipid (PoL) mixture with highpressure/high temperature (HP/HT) supercritical carbon dioxide (SCCO2).Extraction can be performed by any method known in the art, includingbatchwise and continuous flow methods (e.g., countercurrent columns). Wehave found that SCCO2 extracts neutral lipids completely withessentially zero polar lipids in either the form of phospholipids orglycolipids. SCCO2 in the range from 100 to 1000 bar, e.g., 300 to 1000bar, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 bar, andtemperatures between 35 and 110° C., e.g., 60 and 110° C., e.g., 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 110° C., has a highdistribution coefficient for neutral lipids and, essentially, a zerodistribution coefficient for polar lipids. In varying embodiments, theCAO is contacted with SCCO2 at pressures in the range of about 340 barto about 700 bar and at temperatures in the range of about 40° C. toabout 110° C. In varying embodiments, the CAO is contacted with SCCO2 atpressures in the range of about 350 bar to about 690 bar and attemperatures in the range of about 60° C. to about 90° C. At 350 bar and60° C., the density of SCCO2 is 0.863 g/mL. At 700 bar/100° C., SCCO2has a density of 0.9 g/mL. Process conditions in the pressure rangebetween 340 bar and 700 bar that yield a density of 0.83 to 0.9 g/mL aresuitable. High P/T SCCO2 produces a NL fraction with zero PoL. Itextracts a proportion of the chlorophyll and almost all the sterols fromthe CAO. The NL fraction is comprised of free fatty acids (FFA),triglycerides (TG), diglycerides (DG), chlorophyll, and sterols. Theresidual material from high P/T SCCO2 extraction is concentrated polarlipids (Conc PoL), including phosopholipids and glycolipids. The ConcPoL is the second component in the EPA-standardized blend. This streamand the COA provide the polar lipids for the EPA standardized EPA/PolarLipid blend.

In alternative embodiment, CAE can be split into an NL rich fraction andPoL rich fraction using HP/HT SCCO2 followed by extraction with dimethylether (DME).

e. Isolating C20 Free Fatty Acids from the Neutral Lipid Fraction

The NL-rich fraction of the CAO, a portion of the total CAO or a portionof the CAE is hydrolyzed to form free fatty acid (FFA) and then subjectto SCCO2 fractionation to create a concentrate of EPA FFA. In varyingembodiments, a first portion of the CAE, e.g., 30-80% of the total CAE,e.g., 30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, or 80% of theCAE is directly subject to hydrolysis. The second portion of the CAE canbe reserved to include in the final blended EPA formulation.

Hydrolysis to release free fatty acids can be done by a variety ofroutes that are familiar to those in the art. The most common methodsare saponification followed by acidification and direct acidification.In terms of product yield, saponification is a useful route because thefirst step in the reaction irreversibly forms a fatty acid salt. Invarying embodiments, the neutral lipid mixture can be combined with ahydroxyl salt, e.g., KOH or NaOH, in the presence of an excess of thewater. The oxyl bond between the fatty acid and the glycerol backbone isbroken and the respective cationic interaction, e.g., K⁺ or Na⁺ cationicinteraction, formed. This reaction can be completed under reflux attemperature conditions ranging between 50° C. and 90° C. Triglyceride(TG) and diglyceride (DG) constituents are converted to a salt and freeglycerol. Free glycerol is highly polar. The salt solution is treatedwith an acid, such as phosphoric, sulfuric, hydrochloric, or formicacid. This removes the salt's cation and forms the corresponding freefatty acid (FFA). The solution partitions into two phases: an organicand aqueous phase. In the direct acidification method, the reaction hasfewer steps but is reversible. Hence, the yield to FFA may not be asgreat as the saponification route. Under acidification, neutral lipid iscombined with water and strong acid, such as sulfuric, hydrochloric,phosphoric, or formic. Water in excess of stoichiometry, e.g., on theorder of at least about 5×, e.g., about 6×, 7×, 8×, 9×, 10×, or more, isadded to the neutral lipid. Acid is added to lower the pH toapproximately 2. The mixture is heated under reflux at a temperaturebetween 60 and 100° C. This reaction, while single step, is reversible.An excess of water is required to drive the equilibrium in the directionof FFA. The mixture of biomass can then be solvent extracted, e.g., withhexanes. After evaporating the solvent, a partially hydrolyzed algae oilis recovered comprised of predominantly fatty acids of which nearly halfare free fatty acids.

Once the neutral lipid has been hydrolyzed to form FFA, the EPA fractionwithin this mixture can be further concentrated and isolated fromshorter chain length fatty acids. Under the previous processing step,all the triglycerides and diglycerides have been converted to FFA. Thisis known as high acid oil, a mixture of different fatty acid (FA)compounds that are predominantly in free fatty acid form. While is itknown from the literature that SCCO2 can concentrate Omega-3 from methylesters and, by extension, ethyl esters (Nilsson, et. al., “SupercriticalFluid CO2 Fractionation of Fish Oil Esters” in Advances in SeafoodBiochemistry, 1992), it was not previously known that SCCO2 couldfractionate mixtures of non-esterified FFA (FFA FA). FFA FA are polarmoieties. Conventional thought in SCCO2 solubility is that thesecompounds would be insoluble in SCCO2 and, thus, not be amenable totunable dissolving characteristics of SCCO2. Surprisingly, we have foundthat SCCO2 is capable of fractionating FFA FA by molecular weight. TheFFA FA can be fractionated by applying a pressure or temperaturegradient.

In varying embodiments, the FFA FA feedstock is fractionated under apressure gradient of SCCO2. Without being bound by any particulartheory, the non-polar effect of long carboxylic acid chain from 8 to 20carbon molecules long overwhelms the polar characteristics of thecarbonyl group. Thus, in the presence of isothermal conditions,increasing SCCO2 pressure from about 100 bar results in increasinglygreater solubility for higher molecular weight carboxylic acids. Lowerpressure SCCO2 at pressures above 100 bar, e.g., a stepwise orcontinuous gradient over pressures in the range of about 150 bar toabout 350 bar, under isothermal conditions, e.g., at a temperature inthe range of about 40° C. to about 60° C., can be used to remove thelower molecular weight free fatty acids from the higher molecular weightfree fatty acids. This enables concentrating the C20 components,including EPA and ARA, while reducing or eliminating the C8, C10, C12,C14, C18 constituents. In varying embodiments, the EPA concentration isat least doubled. After concentration, this is the EPA-Concentrated FFAsteam (Conc EPA) and is the third constituent in the mixture to createan EPA-standardized formulation.

f. Combining the C20 Free Fatty Acid Fraction and the Polar LipidFraction

Three components are blended to form a standardized combination of EPAand polar lipids: CAO, Conc PoL, and Conc EPA are used to create astandardized product that controls both the EPA and the polar lipidcontent in the blend.

4. Methods of Preventing and Treating Conditions Mitigated by EPA

Eicosapentaenoic acid (EPA, C20:5, n-3) is an important fatty acid inthe omega-3 family based on its medically established therapeuticcapabilities against numerous disease conditions and disorders,including without limitation psychiatric disorders (e.g., depression(including major depression, depressed mood and/or post-partumdepression), bipolar disorder, anxiety, panic and social phobicdisorders, mood disorders, schizophrenia, Obsessive Compulsive Disorder(OCD), borderline personality disorder, attention deficit hyperactivitydisorder and related disorders, anorexia nervosa), cardiovasculardiseases, osteopathic disorders (e.g., osteoarthritis, osteoporosis),cancers, and neurodegenerative disorders (e.g., Alzheimer's disease,Parkinson's disease, dementia, Huntington's disease, amyotrophic lateralsclerosis or any other “triplet repeat” disease, stroke, multi-infarctor other form of dementia, multiple sclerosis, chronic fatigue andepilepsy). See, e.g., Hegarty, et al., Curr Opin Psychiatry. (2013)26(1):33-40; Parker, et al., Am J Psychiatry. (2006) 163(6):969-78;Martins, J Am Coll Nutr. (2009) 28(5):525-42; Stahl, et al., Curr OpinInvestig Drugs. (2008) 9(1):57-64; Simopoulos, Am. J Clin. Nutr. (1999)70:560S-569S; and Ursin. J Nutr. (2003) 133:4271-4274). The EPAformulations described herein find use in the prevention, amelioration,mitigation, delay of progression of, and/or treatment of any diseasecondition found to be prevented, ameliorated, mitigated, delayed and/ortreated by EPA.

a. Subjects who may Benefit

Subjects/patients amenable to prevention, amelioration, mitigation,delay of progression of, and/or treatment by administration of aneffective amount of the EPA compositions described herein includeindividuals at risk of disease but not showing symptoms, as well assubjects presently showing symptoms. In certain embodiments, aneffective amount of the EPA formulations are administered to individualswho do have a known genetic risk of the disease condition, whether theyare asymptomatic or showing symptoms of disease. Such individualsinclude those having relatives who have experienced or been diagnosedwith the disease condition (e.g., a parent, a grandparent, a sibling),and those whose risk is determined by analysis of genetic or biochemicalmarkers. In some embodiments, the subject is asymptomatic but hasfamilial and/or genetic risk factors for developing the diseasecondition. In some embodiments, the subject is exhibiting symptoms ofdisease or has been diagnosed as having the disease condition.

b. Conditions Amenable to Treatment

The EPA formulations described herein can be used for the prevention,amelioration, mitigation, delay of progression of, and/or treatment of awide range of diseases and disorders including without limitation: anypsychiatric, neurological or other central or peripheral nervous systemdisease—in particular depression, schizophrenia, bipolar disorder,anorexia nervosa and degenerative disorders of the brain includingAlzheimer's disease and other dementias and Parkinson's disease; asthmaand other respiratory diseases; inflammatory disease affecting anysystem; any form of inflammatory disease including any form ofarthritis, any form of inflammatory skin disease including psoriasis andeczema, any form of inflammatory gastrointestinal disease includingulcerative colitis, Crohn's disease, inflammatory bowel diseases,irritable bowel syndrome, and any inflammatory conditions of any otherorgans including the eyes and brain; any form of cardiovascular orcerebrovascular disease; any form of metabolic disease includingdiabetes, syndrome X, and any disturbance of calcium metabolismincluding osteoporosis, unolithiase, or urinary tract stone formation;any form of renal or urinary tract disease; any form of disease ordisorder of the reproductive system or menstrual cycle; kidney orurinary tract diseases; liver diseases; disease of the male or femalereproductive organs such as the breast or the prostate gland; cancerand/or cancer cachexia; diseases of the head and neck, including diseaseof the mouth and teeth, of the eyes or of the ears; infection withviruses, bacteria, fungi, protozoa or other organisms.

Illustrative disease conditions that can be prevented, ameliorated,mitigated, delayed and/or treated by administration of an effectiveamount of the EPA compositions described herein include withoutlimitation psychiatric disorders (e.g., depression (including unipolardepression, major depression, depressed mood and/or post-partumdepression), bipolar disorder, mood disorders, schizophrenia,schizoaffective disorders, schizotypy, borderline personality disorder,attention deficit hyperactivity disorder and related disorders. anorexianervosa), osteopathic disorders (e.g., osteoarthritis, osteoporosis),cardiovascular diseases (e.g., hypertension, coronary artery disease,hypercholesterolemia, dyslipidaemia, high blood pressure, and peripheralvascular system disease), cancers, cancer cachexia, neurodegenerativedisorders (e.g., Alzheimer's disease, Parkinson's disease, dementia,Huntington's disease, amyotrophic lateral sclerosis or any other“triplet repeat” disease, stroke, multi-infarct or other form ofdementia, multiple sclerosis, chronic fatigue and epilepsy), asthma andother respiratory diseases, liver diseases (e.g., chronic hepatitis;steatosis; liver fibrosis; cirrhosis), alcoholism; malnutrition; chronicparenteral nutrition; phospholipid deficiency; lipid peroxidation;disarrhythmia of cell regeneration; destabilization of cell membranes;menopausal or post-menopausal conditions; aging; benign prostatichyperplasia; kidney disease; edema; skin diseases; gastrointestinaldiseases (e.g., inflammatory bowel diseases and irritable bowelsyndrome); and pregnancy toxemia. In varying embodiments, the EPAformulations can be taken as a general nutritional supplement.

Accordingly, methods of preventing, ameliorating, mitigating, delayingof progression of, and/or treating any of the aforesaid diseases orconditions, in particular neurological and psychiatric disorders, e.g.,schizophrenia, schizoaffective disorders, schizotypy, depression(including major depression, depressed mood and/or post-partumdepression), bipolar disorder, mood disorders, schizophrenia, borderlinepersonality disorder, attention deficit hyperactivity disorder andrelated disorders by administration of an effective amount of the EPAcompositions described herein are provided.

Furthermore, methods of preventing, ameliorating, mitigating, delayingof progression of, and/or treating any disease selected from: asthma andother respiratory diseases; degenerative disorders of the brainincluding Alzheimer's disease and other dementias and Parkinson'sdisease; diseases of the gastrointestinal tract including inflammatorybowel diseases and irritable bowel syndrome; inflammatory diseaseaffecting any system; cardiovascular disease; any form of dyslipidaemia,any form of diabetes or any form of metabolic diseases; any form ofdermatological diseases; any form of kidney or urinary tract disease;any form of liver disease; any form of disease of the male or femalereproductive system or related secondary sexual organs such as thebreast or prostate gland; any form of cancer or for cancer cachexia; anydisease of the head and neck including diseases of the mouth and teeth,of the eyes or of the ears; and any form of infection with viruses,bacteria, fungi, protozoa or other organisms by administration of aneffective amount of the EPA compositions described herein are alsoprovided.

c. Formulation and Administration

In one embodiment, the EPA compositions are orally deliverable. Theterms “orally deliverable” or “oral administration” herein include anyform of delivery of a therapeutic agent or a composition thereof to asubject wherein the agent or composition is placed in the mouth of thesubject, whether or not the agent or composition is swallowed. Thus“oral administration” includes buccal and sublingual as well asesophageal administration.

In some embodiments, the EPA compositions are in the form of soliddosage forms. Non-limiting examples of suitable solid dosage formsinclude tablets (e.g. suspension tablets, bite suspension tablets, rapiddispersion tablets, chewable tablets, melt tablets, effervescenttablets, bilayer tablets, etc), caplets, capsules (e.g. a soft or a hardgelatin capsule from animal gelatin or from a vegetarian source filledwith solid and/or liquids), powder (e.g. a packaged powder, adispensable powder or an effervescent powder), lozenges, sachets,cachets, troches, pellets, granules, microgranules, encapsulatedmicrogranules, powder aerosol formulations, or any other solid dosageform reasonably adapted for oral administration.

In varying embodiments, the present EPA compositions can be formulatedin single or separate dosage units. The terms “dose unit” and “dosageunit” herein refer to a portion of a pharmaceutical composition thatcontains an amount of a therapeutic agent suitable for a singleadministration to provide a therapeutic effect. Such dosage units may beadministered one to a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1to 4 or 1 to 2) of times per day, or as many times as needed to elicit apreventative, mitigating and/or therapeutic response.

In another embodiment, the EPA composition(s) can be in the form ofliquid dosage forms or dose units to be imbibed directly or they can bemixed with food or beverage prior to ingestion. Non-limiting examples ofsuitable liquid dosage forms include solutions, suspension, elixirs,syrups, liquid aerosol formulations, etc. Generally, the liquid formsare not biphasic and contain less than about 10 wt. % H₂O, e.g., lessthan about 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt.%, 2 wt. %, or 1 wt. % H₂O.

In varying embodiments, the EPA compositions are formulated foradministration of EPA at a daily dose of less than or equal to 2 grams,e.g., less than 1 gram, less than 100 mg, less than 10 mg, less than 1mg, e.g., about 1 mg to about 10 mg, e.g., about 10 mg to about 100 mg,e.g., about 10 mg to about 2 g, e.g., about 100 mg to about 2 g. Invarying embodiments, the EPA compositions are formulated foradministration of EPA at a total dosage in the range of 250 mg to 2 gper day. For example, the EPA compositions may be formulated foradministration of EPA at a dose of 60 to 100 mg in a 300 mg capsule,e.g., 90 to 170 mg in a 500 mg capsule, e.g., 180 to 340 mg in a 1000 mgcapsule.

In some embodiments, the EPA compositions further comprise a stabilizingagent that suppresses, prevents, hinders, or otherwise attenuates thedecomposition of the active ingredient(s) during storage. For example,oxidative decomposition of EPA in compositions may be prevented orattenuated by the presence of antioxidants. Non-limiting examples ofsuitable antioxidants include tocopherol, Origanox™ (available fromFrutarom Ltd.), lecithin, citric acid and/or ascorbic acid. One or moreantioxidants, if desired, are typically present in a composition in anamount of about 0.001% to about 5%, about 0.005% to about 2.5%, or about0.01% to about 1%, by weight.

Excipients

The EPA compositions optionally can comprise one or morepharmaceutically acceptable excipients. The term “pharmaceuticallyacceptable excipient” herein means any substance, not itself atherapeutic agent, used as a carrier or vehicle for delivery of atherapeutic agent to a subject or added to a pharmaceutical compositionto improve its handling or storage properties or to permit or facilitateformation of a unit dose of the composition, and that does not produceunacceptable toxicity or interaction with other components in thecomposition.

The EPA compositions optionally can comprise one or morepharmaceutically acceptable diluents as excipients. Suitable diluentsillustratively include, either individually or in combination, lactose,including anhydrous lactose and lactose monohydrate; starches, includingdirectly compressible starch and hydrolyzed starches (e.g., Celutab™ andEmdex™); mannitol; sorbitol; xylitol; dextrose (e.g., Cerelose™ 2000)and dextrose monohydrate; dibasic calcium phosphate dihydrate;sucrose-based diluents; confectioner's sugar; monobasic calcium sulfatemonohydrate; calcium sulfate dihydrate; granular calcium lactatetrihydrate; dextrates; inositol; hydrolyzed cereal solids; amylose;celluloses including microcrystalline cellulose, food grade sources ofa- and amorphous cellulose (e.g., Rexcel™) and powdered cellulose;calcium carbonate; glycine; bentonite; polyvinylpyrrolidone; and thelike. Such diluents, if present, can constitute in total about 5% toabout 99%, about 10% to about 85%, or about 20% to about 80%, of thetotal weight of the composition.

The EPA compositions optionally can comprise one or morepharmaceutically acceptable disintegrants as excipients. Suitabledisintegrants include, either individually or in combination, starches,including sodium starch glycolate (e.g., Explotab™ of PenWest) andpregelatinized corn starches (e.g., National™ 1551, National™ 1550, andColocorn™ 1500), clays (e.g., Veegum™ HV), celluloses such as purifiedcellulose, microcrystalline cellulose, methylcellulose,carboxymethylcellulose and sodium carboxymethylcellulose, croscarmellosesodium (e.g., Ac-Di-Sol™ of FMC), alginates, crospovidone, and gums suchas agar, guar, xanthan, locust bean, karaya, pectin and tragacanth gums.Such disintegrants, if present, typically comprise in total about 0.2%to about 30%, about 0.2% to about 10%, or about 0.2% to about 5%, of thetotal weight of the composition.

The EPA compositions optionally can comprise one or more antioxidants.Illustrative antioxidants include sodium ascorbate, Origanox™, andvitamin E (tocopherol). One or more antioxidants, if present, aretypically present in the EPA composition in an amount of about 0.001% toabout 5%, about 0.005% to about 2.5%, or about 0.01% to about 1%, byweight.

The EPA compositions optionally can comprise one or morepharmaceutically acceptable binding agents or adhesives as excipients.Such binding agents and adhesives can impart sufficient cohesion to apowder being tableted to allow for normal processing operations such assizing, lubrication, compression and packaging, but still allow thetablet to disintegrate and the composition to be absorbed uponingestion. Suitable binding agents and adhesives include, eitherindividually or in combination, acacia; tragacanth; sucrose; gelatin;glucose; starches such as, but not limited to, pregelatinized starches(e.g., National™ 1511 and National™ 1500); celluloses such as, but notlimited to, methylcellulose and carmellose sodium (e.g., Tylose™);alginic acid and salts of alginic acid; magnesium aluminum silicate;PEG; guar gum; polysaccharide acids; bentonites; povidone, for examplepovidone K-15, K-30 and K-29/32; polymethacrylates; HPMC;hydroxypropylcellulose (e.g., Klucel™); and ethylcellulose (e.g.,Ethocel™). Such binding agents and/or adhesives, if present, constitutein total about 0.5% to about 25%, about 0.75% to about 15%, or about 1%to about 10%, of the total weight of the composition.

The EPA compositions optionally can comprise one or morepharmaceutically acceptable wetting agents as excipients. Non-limitingexamples of surfactants that can be used as wetting agents in the EPAcompositions include quaternary ammonium compounds, for examplebenzalkonium chloride, benzethonium chloride and cetylpyridiniumchloride, dioctyl sodium sulfosuccinate, polyoxyethylene alkylphenylethers, for example nonoxynol 9, nonoxynol 10, and octoxynol 9,poloxamers (polyoxyethylene and polyoxypropylene block copolymers),polyoxyethylene fatty acid glycerides and oils, for examplepolyoxyethylene (8) caprylic/capric mono- and diglycerides (e.g.,Labrasol™ of Gattefosse), polyoxyethylene (35) castor oil andpolyoxyethylene (40) hydrogenated castor oil; polyoxyethylene alkylethers, for example polyoxyethylene (20) cetostearyl ether,polyoxyethylene fatty acid esters, for example polyoxyethylene (40)stearate, polyoxyethylene sorbitan esters, for example polysorbate 20and polysorbate 80 (e.g., Tween™ 80 of ICI), propylene glycol fatty acidesters, for example propylene glycol laurate (e.g., Lauroglycol™ ofGattefosse), sodium lauryl sulfate, fatty acids and salts thereof, forexample oleic acid, sodium oleate and triethanolamine oleate, glycerylfatty acid esters, for example glyceryl monostearate, sorbitan esters,for example sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate and sorbitan monostearate, tyloxapol, and mixturesthereof. Such wetting agents, if present, constitute in total about0.25% to about 15%, about 0.4% to about 10%, or about 0.5% to about 5%,of the total weight of the composition.

The EPA compositions optionally can comprise one or morepharmaceutically acceptable lubricants (including anti-adherents and/orglidants) as excipients. Suitable lubricants include, eitherindividually or in combination, glyceryl behapate (e.g., Compritol™888); stearic acid and salts thereof, including magnesium (magnesiumstearate), calcium and sodium stearates; hydrogenated vegetable oils(e.g., Sterotex™); colloidal silica; talc; waxes; boric acid; sodiumbenzoate; sodium acetate; sodium fumarate; sodium chloride; DL-leucine;PEG (e.g., Carbowax™ 4000 and Carbowax™ 6000); sodium oleate; sodiumlauryl sulfate; and magnesium lauryl sulfate. Such lubricants, ifpresent, constitute in total about 0.1% to about 10%, about 0.2% toabout 8%, or about 0.25% to about 5%, of the total weight of thecomposition.

Suitable anti-adherents include talc, cornstarch, DL-leucine, sodiumlauryl sulfate and metallic stearates. Talc is an anti-adherent orglidant used, for example, to reduce formulation sticking to equipmentsurfaces and also to reduce static in the blend. Talc, if present,constitutes about 0.1% to about 10%, about 0.25% to about 5%, or about0.5% to about 2%, of the total weight of the composition. Glidants canbe used to promote powder flow of a solid formulation. Suitable glidantsinclude colloidal silicon dioxide, starch, talc, tribasic calciumphosphate, powdered cellulose and magnesium trisilicate.

Compositions of the present invention optionally comprise one or moreflavoring agents, sweetening agents, and/or colorants. Flavoring agentsuseful in the present invention include, without limitation, acaciasyrup, alitame, anise, apple, aspartame, banana, Bavarian cream, berry,black currant, butter, butter pecan, butterscotch, calcium citrate,camphor, caramel, cherry, cherry cream, chocolate, cinnamon, citrus,citrus punch, citrus cream, cocoa, coffee, cola, cool cherry, coolcitrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose,fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup,grape, grapefruit, honey, isomalt, lemon, lime, lemon cream,MagnaSweet®, maltol, mannitol, maple, menthol, mint, mint cream, mixedberry, nut, orange, peanut butter, pear, peppermint, peppermint cream,Prosweet® Powder, raspberry, root beer, rum, saccharin, safrole,sorbitol, spearmint, spearmint cream, strawberry, strawberry cream,stevia, sucralose, sucrose, Swiss cream, tagatose, tangerine, thaumatin,tutti fruitti, vanilla, walnut, watermelon, wild cherry, wintergreen,xylitol, and combinations thereof, for example, anise-menthol,cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint,honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream,vanilla-mint, etc.

Sweetening agents that can be used in the present invention include, forexample, acesulfame potassium (acesulfame K), alitame, aspartame,cyclamate, cylamate, dextrose, isomalt, MagnaSweet®, maltitol, mannitol,neohesperidine DC, neotame, Prosweet® Powder, saccharin, sorbitol,stevia, sucralose, sucrose, tagatose, thaumatin, xylitol, and the like.

Flavoring agents, sweetening agents, and/or colorants can be present inthe EPA compositions in any suitable amount, for example about 0.01% toabout 10%, about 0.1% to about 8%, or about 1% to about 5%, by weight.

The EPA compositions optionally can comprise a suspending agent.Non-limiting illustrative examples of suitable suspending agents includesilicon dioxide, bentonite, hydrated aluminum silicate (e.g. kaolin) andmixtures thereof. One or more suspending agents are optionally presentin the EPA compositions in a total amount of about 0.01% to about 3.0%,about 0.1% to about 2.0%, or about 0.25% to about 1.0%, by weight

The foregoing excipients can have multiple roles as is known in the art.For example, starch can serve as a filler as well as a disintegrant. Theclassification of excipients above is not to be construed as limiting inany manner. Excipients categorized in any manner may also operate undervarious different categories of excipients as will be readilyappreciated by one of ordinary skill in the art.

When the EPA compositions are formulated as nutraceuticals, they can bein the form of foods, beverages, energy bars, sports drinks, supplementsor other forms all as are known in the art.

Combination Therapies

In varying embodiments, the EPA formulations can be co-administered withan antidepressant, an antihypertensive agent and/or a cholesterolreducing agent, astaxanthin, vitamin E, phospholipids, coenzyme Q9(CoQ9), and/or coenzyme Q10 (CoQ10). Co-administration with the hereindescribed EPA formulations can allow for administration of theantidepressant, antihypertensive agent and/or cholesterol reducing agentat a subtherapeutic dose.

Illustrative antidepressants that can be co-administered with thepresent EPA formulations include without limitation, selective serotoninreuptake inhibitors, SSRIs (e.g., citalopram, escitalopram, paroxetine,fluoxetine, fluvoxamine, sertraline); selective norepinephrine reuptakeinhibitors (NRIs) (e.g., atomoxetine, reboxetine, viloxazine);noradrenergic and specific serotonergic antidepressants (NaSSA) (e.g.,mianserin, mirtazapine); serotonin-norepinephrine reuptake inhibitors(SNRIs) (e.g., desvenlafaxine, duloxetine, milnacipran, venlafaxine);serotonin antagonist and reuptake inhibitors (SARIs) (e.g., etoperidone,nefazodone, trazodone); norepinephrine-dopamine reuptake inhibitors(e.g., bupropion); selective serotonin reuptake enhancers (e.g.,tianeptine, amineptine); norepinephrine-dopamine disinhibitors (NDDIs)(e.g., agomelatine); tricyclic antidepressants (e.g., amitriptyline,clomipramine, doxepin, imipramine, trimipramine, desipramine,nortriptyline, protriptyline); monoamine oxidase inhibitors (MAOIs)(e.g., isocarboxazid, moclobemide, phenelzine, pirlindole, selegiline,tranylcypromine).

Illustrative antihypertensive agents that can be co-administered withthe present EPA formulations include without limitation, loop diuretics(e.g., bumetanide, ethacrynic acid, furosemide, torsemide); thiazidediuretics (e.g., epitizide, hydrochlorothiazide, chlorothiazide,bendroflumethiazide); thiazide-like diuretics (e.g., indapamide,chlorthalidone, metolazone); potassium-sparing diuretics (e.g.,amiloride, triamterene, spironolactone); beta adrenergic receptorblockers (e.g., atenolol, metoprolol, nadolol, nebivolol, oxprenolol,pindolol, propranolol, timolol); alpha adrenergic receptor blockers(e.g., doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin,terazosin, tolazoline); mixed alpha+beta blockers (e.g., bucindolol,carvedilol, labetalol); calcium channel blockers (e.g., amlodipine,felodipine, isradipine, lercanidipine, nicardipine, nifedipine,nimodipine, nitrendipine, diltiazem, verapamil); renin inhibitors (e.g.,aliskiren); angiotensin-converting enzyme (ACE) inhibitors (e.g.,captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril,ramipril, trandolapril, benazepril); angiotensin II receptor antagonists(e.g., candesartan, eprosartan, irbesartan, losartan, olmesartan,telmisartan, valsartan); aldosterone receptor antagonists (e.g.,eplerenone, spironolactone); vasodilators (e.g., sodium nitroprusside,hydralazine); alpha-2 agonists (e.g., clonidine, guanabenz, methyldopa,moxonidine) and adrenergic neuron blockers (e.g., guanethidine,reserpine).

Illustrative hypolipidemic agents (a.k.a., antihyperlipidemic agents orlipid lowering drugs) that can be co-administered with the present EPAformulations include without limitation, statins or HMG-CoA reductaseinhibitors (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin),fibrates (e.g., bezafibrate, ciprofibrate, clofibrate, gemfibrozil,fenofibrate), niacin, bile acid sequestrants (resins) (e.g.,cholestyramine, colesevelam, colestipol, colestipid), ezetimibe,lomitapide, phytosterols (e.g., β-sitosterol, campesterol,stigmasterol), and orlistat.

The EPA formulations can be co-administered with a therapeuticallyeffective amount or a sub-therapeutic amount of one or more of anantidepressant, antihypertensive agent and/or antihyperlipidemic agent.The dosage of the specific compounds depends on many factors that arewell known to those skilled in the art. They include for example, theroute of administration and the potency of the particular compound.Dosing and scheduling of antidepressants, antihypertensive agents and/orantihyperlipidemic agents are known in the art, and can be found, e.g.,in the published literature and in reference texts, e.g., thePhysicians' Desk Reference, 67th Ed., 2013, Thomson Healthcare orBrunton, et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 12th edition, 2010, McGraw-Hill Professional). Because ofthe cooperative action between the EPA formulations and theantidepressants, antihypertensive agents and/or antihyperlipidemicagents, one or both of the co-administered agents can be administered ata sub-therapeutic dose.

Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. Generally, an efficacious or effective amount of acombination of one or more polypeptides of the present invention isdetermined by first administering a low dose or small amount of apolypeptide or composition and then incrementally increasing theadministered dose or dosages, adding a second or third medication asneeded, until a desired effect of is observed in the treated subjectwith minimal or no toxic side effects. Applicable methods fordetermining an appropriate dose and dosing schedule for administrationof a combination of the present invention are described, for example, inGoodman and Gilman's The Pharmacological Basis of Therapeutics, 12thEdition, 2010, supra; in a Physicians' Desk Reference (PDR), 67^(th)Edition, 2013; in Remington: The Science and Practice of Pharmacy,21^(st) Ed., 2005, supra; and in Martindale: The Complete DrugReference, Sweetman, 2005, London: Pharmaceutical Press., and inMartindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996,Amer Pharmaceutical Assn, each of which are hereby incorporated hereinby reference.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 EPA Formulations with Improved Bioavailability

This Standardized Omega 3 and Polar Lipid Formulation derives from twostrains of the microalgae Nannochloropsis oculata, hereafter referred toas S12 and S14. N. Oculata is a marine algal strain and, thus, must begrown in either seawater or brackish water. Brackish water has betweenone fifth and one times the dissolved solids that are present inseawater. Neither S12 nor S14 have been genetically modified. Thestrains are a result of selective breeding program. The S12 strain is,nominally, adapted for lower ambient temperature conditions, while theS14 can grow in warm temperature conditions. There is a natural variancein the composition of the algae due to variety of factors that includebut are not limited to strain, media content, diurnal temperaturevariation, illumination, culture concentration.

In addition, the extract composition is a function of the handling ofthe algal biomass upon its removal from the growth system. Nominally,the algae grows in relatively dilute culture on a system that istypically on the range of 0.1 to 1.0 g/L of biomass and, more typically,in the range of 0.4 to 0.7 g/L. For a 0.5 g/L culture concentration,this implies that there is 0.5 g of dry weight equivalent biomass forevery 1000 g of culture, a dilute concentration. Algae is furtherprocessed in a more concentrated state, typically in the range of 2 to300 g/L, so a significant amount of water needs to be removed. Water, inthis case, is understood to be saltwater, water with dissolved solids.

Omega-3 in S12 and S14 N. Oculata refers to the eicosapentanoic acid(EPA) (C20:5ω3) and alpha-linolenic acid (ALA) (C18:3ω3), nominallywhere the EPA represented the substantial fraction of the total Omega-3.Polar lipids include both phospholipids (PL) and glycolipids (GL). ThePL fraction is comprised of four PL components: phosphatidylcholine(PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), andphosphatidylinositol (PI). The glycolipids in the mixture arepredominantly digalactosyldiacylglycerol (DGDG) andmonogalactosyldiacylglycerol (MGDG). Omega-7 is nominally represented bypalmitoleic acid (C16:1ω7). The fatty acids (FA) in the mixture areassociated with four major lipid types: PL, GL, free fatty acids (FFA),and triglycerides (TG). There are also minor components of diglycerides(DG) present. Neutral lipids (LP) are comprised of FFA, TG, and DG.Polar lipids (PoL) are comprised of PL and GL.

A specific embodiment of the process to create the standardizedformulation is shown in FIG. 4. This differs from FIG. 1 in the detailsfor Neutral and Polar Lipid Separation (NL/PoL Separation), NeutralLipid Harmonization, and Lipid Fractionation. There are several variantson Biomass Extraction and then specific embodiments for NL/PoLSeparation using supercritical carbon dioxide (SCCO2), Hydrolysis forNeutral Lipid Harmonization, and Lipid Fractionation via SCCO2Fractionation. The Biomass Extraction method is nominally directed tomaximize the recovery of NL and PoL. Because Nannochloropsis Oculata isa phototrophic single cell organism, the photosynthesis mechanisms andthe lipid storage means are located within the same cell. CAE from thebiomass has a high chlorophyll content and can vary from 2 wt % of themixture up to 20 wt %, with a typical value being in the range of 5 to 8wt %. Similarly, sterol content is on the order of 1 wt %. Carotenoidsare between 2,500 and 10,000 ppm (0.25 to 1 wt %).

The Nanno Paste may be extracted in either the wet or dry state. In thewet state, the moisture content is between 400 and 1000% (w/w) driedbiomass (25 to 10 wt % solids). In the dry state, the moisture contentis less than 15% (w/w) of the dried biomass. CAE can contain between 10and 80% of constituents that are not lipids. The components other thanlipids and phytonutrients are termed MONL for Matter Organic-Not Lipid.The composition of this material is not fully known; however, it isknown that the constituents that need to be eliminated are water-solublecomponents. The resultant output of MONL refinement is crude algae oil(CAO) that is in excess of 50 wt % total lipids and, in someembodiments, in excess of 60 or 70 wt % total lipids.

The benchmark technique for determining fatty acid content in algalbiomass is the fat by acid hydrolysis (FAH) method. This involvetreatment of the biomass with a strong acid to digest the cell member,followed by an extraction, conversion to fatty acid methyl esters(FAME), and analysis via AOCS (American Oil Chemist Society) Method Ce1b 89 “Fatty Acid Composition of Marine Oils by GLC” and AOCS Method Ca5b 71 “Crude Fatty Acids”. The former method determines the relativeamount of each fatty acid constituent in the total collection of fattyacids. The latter method determines the total saponifiable fat in asample. The relative amount of each fatty acid normalized by the totalsaponifiable fat determines the sample basis amount of each fatty acid.New Jersey Feed Laboratory, Inc. (NJFL) in Trenton, N.J., USA hasparticular proprietary extensions of the acid digestion, extraction, andFAME conversion. Except as otherwise noted, all FAH profiles aremeasured by this method and organization. Furthermore, where the fattyacid profile (FAP) of a mixture derived via a different extractionmethod, AOCS Methods Ca 5b 71 and Ce 1b 89 are used. Except whereotherwise noted, all FAP data is from NJFL.

The extraction of the lipids and phytonutrients from the remainder ofthe protein, carbohydrate, minerals, and fiber comprising the alga cellsproduces CAE. Biomass extraction results in the lipids being isolatedfrom the biomass while removing a minimal carbohydrates, proteins, andminerals. The residual biomass is substantially depleted of lipids and,thus, termed lipid extracted algae (LEA). Surprisingly and unlike manyother algae species, we have found that N. Oculata can be extractedwithout requiring disruption by either mechanical, thermal, or chemicalmeans to disrupt the cellular membrane. This is illustrated bycomparative extractions of replicate samples (N=3 or 4) S12 and S14biomass in Tables 2 and 3, respectively. All algal biomass was dried ina low humidity environment at 60° C. until the solid was less than 10 wt% moisture. For the fatty acid profiles reported in Tables 2 and 3, thebiomass was processed in a conventional or automated Soxhlet extractor(on the internet at en.wikipedia.org/wiki/Soxhlet_extractor). This isdata reflective of CAE and, thus, has other components other than fatsin the extract. The S12 extract has a lower TFA than the S14 extracts,indicating that there are non-lipid components in this extract. Thesetables illustrate that extraction of dry biomass via FAH and 70/30 v/v %hexane (Hex) and methanol (MeOH) (70/30 Hex/MeOH) solvent extractionthat the extracted EPA on a biomass basis is essentially the same. Thetotal fatty acid (TFA) is sufficiently the same that 70/30 Hex/MeOH canbe applied as a representative and non-proprietary extraction technique.Surprisingly, in S12, the FAH method created an extract higher in TFAthan 70/30 Hex/MeOH while in S14, the 70/30 Hex/MeOH results in thehigher TFA in the extract. Nonetheless, when the amounts are normalizedby the total extract from the biomass, leading to the fatty acid in drysolid, the two methods are produce the nearly the same amount of EPA forboth strains. This is the first of several indications that extractionbehavior of a biomass is very much a function of several variables thatinclude the species, the growth history of the biomass, the harvest andhandling conditions between the time of harvest and the time ofextraction, and solvent system.

The comparison between the fatty acid profile between S12 and S14 ismade in Table 4. This shows dried S12 and S14 processed both by FAH and70/30 Hex/MeOH extraction. In all cases, the saturated FAs are about 25%of the fatty acid profile (FAP), the monosaturated FAs about 30%, andthe polyunsaturated FAs about 35%. The EPA Omega-3 represents greaterthan 98% of the total Omega-3 with both S12 and S14. Thus, the EPA inthe FAP is about 30% and is similar to the Omega-3 in the FAP. S12 has acharacteristic ratio of EPA to ARA of 700 to 900% (i.e. seven to ninetimes the EPA than the ARA). S14 has lower EPA to ARA ratio of greaterthe range of 500 to 600%.

TABLE 2 S12 Extraction of Dry Biomass via Fat by Acid Hydrolysis and70/30 Hex/MeOH Fat by Acid Hydrolysis 70/30 (v/v %) Hexane/MethanolAverage Extract from Biomass % 20.88 36.29 Standard Deviation % 1.664.60 FA in FA in FA in Dry FA in Dry C#:Dbl. Normalized Extract SolidNormalized Extract Solid Fatty Acid Bond FA % % % FA % % % Caprylic  8:00.17 0.11 0.02 0.24 0.11 0.04 Capric 10:0 0.26 0.17 0.04 0.95 0.43 0.16Lauric 12:0 0.45 0.30 0.06 0.38 0.16 0.06 Myristic 14:0 6.66 4.38 0.926.00 2.62 0.95 Myristoleic 14:1 0.67 0.44 0.09 0.09 0.04 0.01Pentadecanoic 15:0 0.43 0.28 0.06 0.41 0.18 0.06 Palmitic 16:0 17.9311.79 2.46 17.17 7.51 2.73 Palmitoleic 16:1 20.83 13.70 2.86 20.55 8.993.26 Hexadecadienoic 16:2 0.20 0.13 0.03 0.19 0.08 0.03 Hexadecatrienoic16:3 0.00 0.00 0.00 0.00 0.00 0.00 Hexadecatetraenoic 16:4 0.08 0.050.01 0.07 0.03 0.01 Heptadecanoic 17:0 0.19 0.13 0.03 0.18 0.08 0.03Stearic 18:0 0.32 0.21 0.04 0.33 0.15 0.05 Oleic 18:1ω9 5.51 3.62 0.765.23 2.29 0.83 Oleic 18:1ω7 0.37 0.24 0.05 0.22 0.10 0.03 Linoleic18:2ω6 4.34 2.85 0.60 4.31 1.88 0.68 Gamma-Linolenic 18:3ω6 0.00 0.000.00 0.20 0.09 0.03 Alpha-Linolenic (ALA) 18:3ω3 0.19 0.13 0.03 0.170.07 0.03 Eicosadienoic 20:2ω6 0.08 0.05 0.01 0.13 0.06 0.02Eicosatrienoic 20:3ω6 0.29 0.19 0.04 0.21 0.09 0.03 Arachidonic 20:4ω63.92 2.58 0.54 3.48 1.52 0.55 Eicosapentaenoic (EPA) 20:5ω3 29.25 19.244.02 27.22 11.90 4.32 Other n/a 7.86 5.17 1.08 12.28 5.38 1.95 TotalFatty Acid 100.00 65.79 13.74 100.00 43.76 15.88 Total Omega-3 29.4419.36 4.04 27.64 16.40 2.89 Total Omega-6 8.63 5.67 1.18 9.08 5.39 0.95

TABLE 3 S14 Extraction of Dry Biomass via Fat by Acid Hydrolysis and70/30 Hex/MeOH Fat by Acid Hydrolysis 70/30 (v/v %) Hexane/MethanolAverage Extract from Biomass % 17.64 16.78 Standard Deviation % 0.561.36 FA in FA in FA in Dry FA in Dry C#:Dbl. Normalized Extract SolidNormalized Extract Solid Fatty Acid Bond FA % % % FA % % % Caprylic  8:00.23 0.13 0.02 0.30 0.23 0.04 Capric 10:0 0.20 0.12 0.02 0.58 0.44 0.07Lauric 12:0 0.49 0.29 0.05 0.35 0.27 0.05 Myristic 14:0 4.39 2.61 0.463.58 2.74 0.46 Myristoleic 14:1 1.06 0.63 0.11 0.12 0.10 0.02Pentadecanoic 15:0 0.34 0.20 0.04 0.34 0.26 0.04 Palmitic 16:0 18.2910.86 1.92 15.91 12.18 2.04 Palmitoleic 16:1 21.41 12.70 2.24 19.1914.69 2.47 Hexadecadienoic 16:2 0.17 0.10 0.02 0.17 0.13 0.02Hexadecatrienoic 16:3 0.04 0.02 0.00 0.18 0.13 0.02 Hexadecatetraenoic16:4 0.15 0.09 0.02 0.16 0.12 0.02 Heptadecanoic 17:0 0.26 0.16 0.030.27 0.21 0.04 Stearic 18:0 0.47 0.28 0.05 0.33 0.25 0.04 Oleic 18:1ω94.81 2.86 0.50 4.26 3.27 0.55 Oleic 18:1ω7 1.25 0.74 0.13 1.10 0.85 0.14Linoleic 18:2ω6 3.80 2.26 0.40 3.55 2.73 0.46 Gamma-Linolenic 18:3ω60.40 0.24 0.04 0.25 0.19 0.03 Alpha-Linolenic (ALA) 18:3ω3 0.39 0.240.04 0.41 0.31 0.05 Eicosadienoic 20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00Eicosatrienoic 20:3ω6 0.04 0.02 0.00 0.04 0.03 0.01 Arachidonic 20:4ω64.84 2.87 0.51 4.08 3.13 0.52 Eicosapentaenoic (EPA) 20:5ω3 27.25 16.172.85 23.47 17.97 3.01 Other n/a 9.71 5.80 1.02 21.37 16.51 2.77 TotalFatty Acid 100.00 59.39 10.48 100.00 76.74 12.88 Total Omega-3 27.6416.40 2.89 27.64 16.40 2.89 Total Omega-6 9.08 5.39 0.95 9.08 5.39 0.95

TABLE 4 Fatty Acid Classes in S12 and S14 Dried Biomass by FAH and 70/30MeOH Extraction S12- FAH S12- 70/30 Hex/MeOH S14- FAH S14- 70/30Hex/MeOH FA in FA in FA in FA in Normalized Extract Normalized ExtractNormalized Extract Normalized Extract Fatty Acid Metric FA % % FA % % FA% % FA % % Total Fatty Acid (TFA) 100.00 65.79 100.00 43.76 100.00 59.39100.00 76.74 Total Saturates 26.42 17.38 25.66 11.24 24.67 14.65 21.6616.58 Total Monounsaturates 27.37 18.01 26.09 11.41 28.53 16.93 24.6718.91 Total Polyunsaturates 38.35 25.22 35.97 15.73 37.08 22.01 32.3024.74 Total Omega-3 29.44 19.36 27.39 11.98 27.64 16.40 23.87 18.28Total Omega-6 8.63 5.67 8.33 3.64 9.08 5.39 7.92 6.08 Total C16 39.0425.68 37.97 16.61 40.06 23.77 35.60 27.26 ARA (20:4ω6) 3.92 2.58 3.481.52 4.84 2.87 4.08 3.13 EPA (20:5ω3) 29.25 19.24 27.22 11.90 27.2516.17 23.47 17.97 Saturates/TFA (%) 26.4 25.7 24.7 21.6Monounsaturates/TFA (%) 27.4 26.1 28.5 24.6 Polyunsaturates/TFA (%) 38.336.0 37.1 32.2 Omega-3/TFA (%) 29.4 27.4 27.6 23.8 EPA/TFA (%) 29.2 27.227.2 23.4 EPA/Total Omega3 (%) 99.4 99.4 98.6 98.3 EPA/ARA (%) 746 781563 575

When the wet or dried Nanno Paste is extracted, cellular membranedisruption is not required to remove the lipids from the biomass. Wetextraction of biomass requires a pure solvent or solvent mixture that isat least partially miscible with water. This includes a broad selectionof solvents types, including ethers, ketones, and alcohols. Some examplesolvents systems are ethanol, isopropyl alcohol, acetone and ethanol,dimethyl ether, dimethyl ether and ethanol. These techniques can besystematically compared using a common feedstock of wet paste (˜15-25 wt%). Furthermore, surprisingly, we have found that biomass extractionrequires no mechanical cracking (such as a bead mill), thermalpretreatment, or cellular wall digestion via acid or base. Theextraction method acts on wet Nanno Paste (˜15-25 wt %), does notutilize any mechanical cracking, thermal pretreatment, or alkaline oracid treatment. In varying embodiments, the solvent system is either anether and alcohol mixture or a ketone and alcohol mixture. Solventpercolation through the biomass paste can be problematic. This can bealleviated by mixing paste with a filtration aid such as Celite®(diatomaceous earth) or Cellu-Flo™ or by vigorous mixing with solventcoupled with crossflow filtration. Example solvent combinations areabsolute ethanol, 190 proof (95 v/v %) ethanol (EtOH), denatured 190proof ethanol, special denatured alcohols (SDA), acetone and ethanol,isopropyl alcohol, acetone and methanol, methyl ethyl ketone (MEK) andmethanol, MEK and ethanol, dimethyl ether, dimethyl ether and methanol,dimethyl ether and ethanol. The common characteristic of the solventmixture is an ability to extract hydrophobic, non-polar lipid componentssuch as triglycerides and hydrophilic, polar lipid components such asphospholipids and glycolipids.

In varying embodiments, the solvent mixture is 50% (v/v) acetone and 50%(v/v) 190 proof ethanol (EtOH). Other example mixtures are pure dimethylether (DME), DME mixed with methanol, or solely 190 proof EtOH. EtOH maybe non-denatured or one of the Special Denatured Alcohol (SDA) grades(1-1, 1-2, 2B-2, 2B-3, 3A, 3C, 23A, 23H, 29, 30, 35A) proof denaturedethanol, where the major composition of the SDAs is given in Table 5. Invarying embodiments, the ethanol is SDA 1-1, 3A, 3C, 23A, or 35A, wherethe major distinguishing characteristics are availability and pricerather than any particular technical advantage for extraction.

TABLE 5 Major Constituents of Special Denatured Alcohols (SDA) EthanolSpecial 190 Denature proof Grade % (v/v) Denaturants SDA 1-1/ 96.15Methanol- 3.85% (v/v) 190 proof SDA 1-2/ 95.24 Methanol- 3.81% (v/v),Methyl Isobutyl 190 proof Ketone (MIBK) - 0.95% (v/v) SDA 2B-2/ 99.50Rubber Solvent- 0.50% (v/v) 190 proof SDA 2B-3/ 99.50 Toluene- 0.50%(v/v) 190 proof SDA 3A 95.24 Methanol- 4.78% (v/v) SDA 3C 95.24Isopropanol- 4.76% (v/v) SDA 23A 92.59 Acetone- 7.41% (v/v) SDA 23H91.32 Acetone- 7.31% (v/v), MIBK- 1.37% (v/v) SDA 29 99.01 Ethylacetate- 0.99% (v/v) SDA 30 90.90 Methanol- 9.10% (v/v) SDA 35A 95.92Ethyl acetate- 4.08% (v/v)

Both S12 and S14 biomass are substantially depleted of lipid by the useof a six stage solvent system. In each stage, two times the mass ofacetone/EtOH mixtures is mixed with the biomass to form a biomass,solvent, and extract slurry. The extract solution is separated from thebiomass by either filtration or centrifugation, where filtration or, insome embodiments, cross-flow filtration is employed for removing solidfrom the solution. For nearly complete lipid extraction, a total of sixstages must be completed. This method creates crude algae extract (CAE).

A comparison of different extraction techniques on dried biomass isgiven in Table 6. In all cases, the cellular material was notmechanically disrupted, thermally pretreated or otherwise subject toalkali or acidic digestion. The biomass was S12 algae grown in northernIsrael. Wet algae concentrated was pooled from multiple harvests overthe summer period, the concentrated wet biomass homogenized, and theresultant biomass solids suspension subjected to spray drying with hotair temperatures of approximately 120° C. The resulting dried algalpowder had a moisture content of less than 10 wt % and, thus, representsalgal biomass with the majority of extracellular and intracellular waterremoved. This dried biomass was extracted in five different ways: FAH byNHL, 70/30 (v/v %) Hex/MeOH by NHL, 50/50 (v/v %) acetone and 190 proofdenatured alcohol, and DME methods. The first DME method involvedpretreatment of spray dried biomass to rewet the algae with a 75/25 (v/v%) mixture of water and methanol (MeOH). The test examined if thebiomass could be rehydrated prior to extraction with DME, a solvent thatis partially miscible with water in the absence of alcohol or ketoneco-solvents (about 6 wt % solubility of water in neat DME). DME ismiscible with a 25 wt % concentration of MeOH in water. The “Wet DME”test involved a light water spray of the dried biomass followed byextract of water-saturated neat DME. In this way, the water acts as aco-solvent for DME.

TABLE 6 Extraction of Spray Dried S12, northern Israel Fat by AcidHydrolysis 70/30 (v/v %) Hex/MeOH 50/50 v/v % acetone/EtOH Test NJFLFAH, 1112-0307 NJFL Hex/MeOH, 1112-0307 QLTS-20 Percent Extract from DryBiomass 13.0 24.7 FA in FA in 30.4 FA in Dry FA in Dry FA in C#:Dbl.Norm. Extract Solid Norm. Extract Solid Norm. Extract Fatty Acid Bond FA% % % FA % % % FA % % Caprylic  8:0 0.29 0.16 0.02 0.42 0.15 0.04 0.380.07 Capric 10:0 0.34 0.19 0.03 0.45 0.16 0.04 0.36 0.07 Lauric 12:00.98 0.55 0.07 0.59 0.21 0.05 0.70 0.14 Myristic 14:0 4.17 2.35 0.313.03 1.07 0.26 3.58 0.71 Myristoleic 14:1 0.74 0.42 0.05 0.00 0.00 0.004.26 0.84 Pentadecanoic 15:0 0.31 0.17 0.02 0.39 0.14 0.03 0.22 0.04Palmitic 16:0 14.95 8.43 1.10 12.23 4.31 1.06 13.12 2.59 Palmitoleic16:1 17.67 9.96 1.29 14.93 5.26 1.30 16.51 3.26 Hexadecadienoic 16:20.42 0.24 0.03 0.38 0.13 0.03 0.34 0.07 Hexadecatrienoic 16:3 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 Hexadecatetraenoic 16:4 0.00 0.00 0.000.28 0.10 0.02 0.00 0.00 Heptadecanoic 17:0 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 Stearic 18:0 0.47 0.27 0.03 0.59 0.21 0.05 0.25 0.05Oleic 18:1ω9 2.95 1.66 0.22 2.21 0.78 0.19 2.77 0.55 Oleic 18:1ω7 1.090.62 0.08 0.92 0.32 0.08 0.88 0.17 Linoleic 18:2ω6 4.48 2.52 0.33 3.381.19 0.29 4.04 0.80 Linoleic 18:2ω4 0.09 0.05 0.01 0.00 0.00 0.00 0.000.00 Gamma-Linolenic 18:3ω6 0.20 0.11 0.01 0.00 0.00 0.00 0.25 0.05Alpha-Linolenic (ALA) 18:3ω3 0.37 0.21 0.03 0.16 0.06 0.01 0.36 0.07Eicosadienoic 20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Eicosatrienoic 20:3ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.02Arachidonic 20:4ω6 4.50 2.54 0.33 4.12 1.45 0.36 3.97 0.78Eicosapentaenoic (EPA) 20:5ω3 35.64 20.09 2.61 35.51 12.52 3.09 35.627.03 Other n/a 10.32 5.82 0.76 20.40 7.19 1.78 12.27 2.42 Total FattyAcid 100.00 56.36 7.33 100.00 35.25 8.71 100.00 19.73 Total Omega-336.01 20.29 2.64 35.67 12.57 3.11 35.97 7.10 Total Omega-6 9.19 5.180.67 7.50 2.64 0.65 8.39 1.66 50/50 v/v % acetone/EtOH H₂O/MeOHPretreat, DME Wet DME Test QLTS-20 QLTS-19 QLTS-18 Percent Extract fromDry Biomass 30.4 19.2 46.0 FA in FA in FA in Dry FA in Dry FA in DryC#:Dbl. Solid Norm. Extract Solid Norm. Extract Solid Fatty Acid Bond %FA % % % FA % % % Caprylic  8:0 0.02 0.44 0.14 0.03 0.38 0.06 0.03Capric 10:0 0.02 0.32 0.10 0.02 0.83 0.12 0.06 Lauric 12:0 0.04 0.880.28 0.06 0.88 0.13 0.06 Myristic 14:0 0.21 3.06 0.96 0.20 3.52 0.520.26 Myristoleic 14:1 0.26 0.08 0.02 0.00 0.00 0.00 0.00 Pentadecanoic15:0 0.01 0.17 0.05 0.01 0.19 0.03 0.01 Palmitic 16:0 0.79 10.94 3.450.72 12.71 1.86 0.93 Palmitoleic 16:1 0.99 14.24 4.49 0.94 15.57 2.281.14 Hexadecadienoic 16:2 0.02 0.33 0.10 0.02 0.45 0.07 0.03Hexadecatrienoic 16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00Hexadecatetraenoic 16:4 0.00 0.00 0.00 0.00 0.32 0.05 0.02 Heptadecanoic17:0 0.00 0.23 0.07 0.01 0.00 0.00 0.00 Stearic 18:0 0.02 0.33 0.10 0.020.47 0.07 0.03 Oleic 18:1ω9 0.17 2.03 0.64 0.13 2.46 0.36 0.18 Oleic18:1ω7 0.05 0.71 0.22 0.05 1.03 0.15 0.08 Linoleic 18:2ω6 0.24 3.36 1.060.22 3.89 0.57 0.28 Linoleic 18:2ω4 0.00 0.00 0.00 0.00 0.00 0.00 0.00Gamma-Linolenic 18:3ω6 0.02 0.24 0.08 0.02 0.00 0.00 0.00Alpha-Linolenic (ALA) 18:3ω3 0.02 0.68 0.21 0.04 0.41 0.06 0.03Eicosadienoic 20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Eicosatrienoic20:3ω6 0.01 0.14 0.04 0.01 0.00 0.00 0.00 Arachidonic 20:4ω6 0.24 4.361.37 0.29 4.14 0.61 0.30 Eicosapentaenoic (EPA) 20:5ω3 2.14 37.14 11.702.44 35.39 5.18 2.59 Other n/a 0.74 20.34 6.41 1.34 17.36 2.54 1.27Total Fatty Acid 6.00 100.00 31.50 6.57 100.00 14.65 7.32 Total Omega-32.16 37.82 11.91 2.49 35.80 5.24 2.62 Total Omega-6 0.50 8.10 2.55 0.538.03 1.18 0.59

As shown in Table 6, the Hex/MeOH results in the greatest amount both ofTFA and Omega 3 of all these methods, as shown by the 8.71% and 3.11% ofthe biomass respectively. For the Hex/MeOH sample, the EPA/Total EPAratio was greater than 99.5% reflective of a very pure S12 sample.Conversely, the FAH method makes the extract most highly concentrated infat, at over 56% versus 35.2, 19.7, 31.5, and 14.6 for the othertechniques. Hex/MeOH results in more recovery of fat and EPA from thedried biomass at the expense of extracting other, non-lipid constituentsthat result in more dilute fatty acid mixture. The two DME methods yieldabout the same amount of fat at 6.57 and 7.32 wt % of biomass andsimilar amounts of EPA at 2.44 and 2.59 wt % of biomass. The H₂O/MeOHversion of DME results in a more highly concentrated extract. Thismethod has a slightly lower yield of EPA from the biomass but yields anextract that is more than twice as concentrated in fatty acid, Omega-3,and EPA. The acetone/EtOH method results in lower recovery of fats,Omega3 and EPA versus the other methods. From the standpoint of driedbiomass, it is the inferior solvent system and, thus, one could concludethat it is not a suitable method. This would fail to account for thebehavior of this solvent system, though, in the presence of a greaterquantity of intracellular and extracellular water.

A good leading indication of the superior benefit of wet extraction overdry extraction can be observed in Table 7. The biomass was nominally ofthe same strain and lot, taken from S12 harvests grown in northernIsrael. The spray dried material was collected over many days while wetslurry reflected a single day's harvest. The wet slurry had a solidscontent of 11.9 wt % or a 98.1 wt % moisture content. The spray driedwas 8 wt % moisture. To put this in perspective, a 100 g of spray driedmaterial was equivalent to 773 g of slurry. This implies that wetextraction, while perhaps more effective in fat and Omega 3 recovery,requires the handling of a significantly higher mass and volume ofbiomass. In the case of wet extraction, the TFA, Omega 3, and EPArecovery on a biomass basis were 18.46, 6.45, and 6.38 wt %,respectively. The same values for the spray dried S12 biomass grown atthe same time were 7.32, 2.62, and 2.59 wt %. Wet extraction yielded250% times the TFA and EPA versus spray dried material. While day-to-dayvariance could perhaps account for a 20 to 50% variance in the TFA andEPA extracted, it was at first implausible to us that the differencewould be this great. Without being bound by any particular theory, thepresence of intracellular water enables EPA and lipids to be extractedthat would otherwise be bound with the dried biomass and, thus, notremoved in the dry state. Wet extraction, also leads to improvedconcentration of fatty acid and EPA in the CAE, with the 30.8 wt % TFAand 10.6 wt % EPA in the CAE versus 14.6 wt % TFA and 5.18 wt % EPA inthe CAE of the spray dried material.

A comparison of different extraction techniques on dried S14 biomass isgiven in Table 8. In all cases, the cellular material was notmechanically disrupted, thermally pretreated or otherwise subject toalkali or acidic digestion. The S14 biomass was grown in northern Israeland subsequently spray dried. All tests were taken from the same lot ofdried biomass. As with the S12, the spray drying was completed with ahot air temperature of approximately 120° C. The resulting dried algalpowder had a moisture content of less than 15 wt %, representing nearlycomplete removal of extracellular and intracellular water. This driedbiomass was extracted in five different ways: 70/30 (v/v %) Hex/MeOH byNJFL, 67/33 (w/w %) Hex/MeOH, acetone, 190 proof (95/5 (v/v %) denaturedEtOH, and dry DME. In the case of the 67/33 Hex/MeOH, acetone and 190proof EtOH, the extraction was performed at room temperature with sixcontact stages, where each stage used two times by weight solvent perunit biomass. Dry DME did not add any water back to the system either bywetting the biomass or by saturating the DME with water. The 70/30 v/v %Hex/MeOH by NJFL used this solvent solution at elevated temperature justbelow the boiling point of the mixture (approximately 60° C.).

TABLE 7 DME Extraction of Dry versus Wet S12 Spray Dried S12- Wet DMEWet S12 Slurry- Dry DME Test QLTS-18 QLTS-17 Percent Extract from DryBiomass 46.0 60.0 FA in FA in FA in Dry FA in Dry C#:Dbl. Norm. ExtractSolid Norm. Extract Solid Fatty Acid Bond FA % % % FA % % % Caprylic 8:0 0.38 0.06 0.03 0.38 0.12 0.07 Capric 10:0 0.83 0.12 0.06 1.29 0.400.24 Lauric 12:0 0.88 0.13 0.06 0.41 0.13 0.08 Myristic 14:0 3.52 0.520.26 3.18 0.98 0.59 Myristoleic 14:1 0.00 0.00 0.00 0.00 0.00 0.00Pentadecanoic 15:0 0.19 0.03 0.01 0.17 0.05 0.03 Palmitic 16:0 12.711.86 0.93 10.61 3.26 1.96 Palmitoleic 16:1 15.57 2.28 1.14 12.28 3.782.27 Hexadecadienoic 16:2 0.45 0.07 0.03 0.65 0.20 0.12 Hexadecatrienoic16:3 0.00 0.00 0.00 0.00 0.00 0.00 Hexadecatetraenoic 16:4 0.32 0.050.02 0.00 0.00 0.00 Heptadecanoic 17:0 0.00 0.00 0.00 0.00 0.00 0.00Stearic 18:0 0.47 0.07 0.03 0.83 0.25 0.15 Oleic 18:1ω9 2.46 0.36 0.183.05 0.94 0.56 Oleic 18:1ω7 1.03 0.15 0.08 0.97 0.30 0.18 Linoleic18:2ω6 3.89 0.57 0.28 3.10 0.95 0.57 Linoleic 18:2ω4 0.00 0.00 0.00 0.000.00 0.00 Gamma-Linolenic 18:3ω6 0.00 0.00 0.00 0.16 0.05 0.03Alpha-Linolenic (ALA) 18:3ω3 0.41 0.06 0.03 0.37 0.11 0.07 Eicosadienoic20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00 Eicosatrienoic 20:3ω6 0.00 0.000.00 0.00 0.00 0.00 Arachidonic 20:4ω6 4.14 0.61 0.30 4.17 1.28 0.77Eicosapentaenoic (EPA) 20:5ω3 35.39 5.18 2.59 34.55 10.63 6.38 Other n/a17.36 2.54 1.27 23.83 7.33 4.40 Total Fatty Acid 100.00 14.65 7.32100.00 30.77 18.46 Total Omega-3 35.80 5.24 2.62 34.91 10.74 6.45 TotalOmega-6 8.03 1.18 0.59 7.43 2.29 1.37

TABLE 8 Spray Dried S14 Extracted by Various Methods 70/30 (v/v %)Hex/MeOH 67/33 w/w Hex/MeOH Acetone Test NJFL Hex/MeOH, 1112-2005QLTS-10 QLTS-11 Percent Extract from Dry Biomass 34.7 11.0 FA in FA in2.7 FA in Dry FA in Dry FA in C#:Dbl. Norm Extract Solid Norm. ExtractSolid Norm. Extract Fatty Acid Bond FA % % % FA % % % FA % % Caprylic 8:0 0.19 0.08 0.027 0.33 0.10 0.011 0.57 0.25 Capric 10:0 0.12 0.050.018 0.25 0.08 0.009 0.31 0.14 Lauric 12:0 0.39 0.16 0.056 0.53 0.170.018 0.76 0.34 Myristic 14:0 4.13 1.73 0.599 4.56 1.43 0.158 4.17 1.86Myristoleic 14:1 0.08 0.03 0.011 0.57 0.18 0.020 0.00 0.00 Pentadecanoic15:0 0.25 0.10 0.036 0.34 0.11 0.012 0.19 0.08 Palmitic 16:0 22.32 9.333.236 21.67 6.79 0.749 14.44 6.45 Palmitoleic 16:1 24.72 10.34 3.58519.53 6.12 0.675 22.77 10.17 Hexadecadienoic 16:2 0.22 0.09 0.032 0.000.00 0.000 0.00 0.00 Hexadecatrienoic 16:3 0.11 0.05 0.016 0.11 0.030.004 0.00 0.00 Heptadecanoic 17:0 0.34 0.14 0.050 0.31 0.10 0.011 0.190.08 Stearic 18:0 0.79 0.33 0.115 0.84 0.26 0.029 0.57 0.25 Oleic 18:1ω93.12 1.30 0.452 5.56 1.74 0.192 5.55 2.48 Oleic 18:1ω7 0.42 0.18 0.0611.60 0.50 0.055 1.16 0.52 Linoleic 18:2ω6 2.93 1.23 0.425 4.07 1.270.141 4.36 1.95 Linoleic 18:2ω4 0.00 0.00 0.000 0.00 0.00 0.000 0.000.00 Gamma-Linolenic 18:3ω6 0.95 0.40 0.137 0.25 0.08 0.009 0.00 0.00Alpha-Linolenic (ALA) 18:3ω3 0.88 0.37 0.128 0.57 0.18 0.020 0.43 0.19Eicosadienoic 20:2ω6 0.00 0.00 0.000 0.00 0.00 0.000 0.00 0.00Eicosatrienoic 20:3ω6 0.33 0.14 0.047 0.00 0.00 0.000 0.00 0.00Arachidonic 20:4ω6 6.05 2.53 0.878 3.23 1.01 0.112 4.15 1.85Eicosapentaenoic (EPA) 20:5ω3 21.33 8.92 3.092 17.32 5.43 0.599 23.4110.45 Other n/a 10.34 4.32 1.499 18.38 5.76 0.635 16.77 7.49 Total FattyAcid 100.00 41.81 14.50 100.00 31.33 3.46 99.81 44.56 Total Omega-322.21 9.29 3.22 17.90 5.61 0.62 23.84 10.64 Total Omega-6 10.26 4.291.49 7.54 2.36 0.26 8.52 3.80 Acetone 95/5 (v/v %) EtOH DME Test QLTS-11QLTS-12 QLTS-8 Percent Extract from Dry Biomass 2.7 23.2 10.5 FA in FAin FA in Dry FA in Dry FA in Dry C#:Dbl. Solid Norm. Extract Solid Norm.Extract Solid Fatty Acid Bond % FA % % % FA % % % Caprylic  8:0 0.0070.30 0.05 0.013 0.45 0.22 0.023 Capric 10:0 0.004 0.18 0.03 0.008 0.890.44 0.046 Lauric 12:0 0.009 0.60 0.11 0.025 0.68 0.33 0.035 Myristic14:0 0.050 4.79 0.87 0.201 5.04 2.46 0.258 Myristoleic 14:1 0.000 0.240.04 0.010 0.08 0.04 0.004 Pentadecanoic 15:0 0.002 0.30 0.05 0.013 0.290.14 0.015 Palmitic 16:0 0.174 22.42 4.05 0.942 20.84 10.19 1.065Palmitoleic 16:1 0.274 21.03 3.80 0.883 18.40 8.99 0.940 Hexadecadienoic16:2 0.000 0.14 0.03 0.006 0.28 0.14 0.014 Hexadecatrienoic 16:3 0.0000.00 0.00 0.000 0.14 0.07 0.007 Heptadecanoic 17:0 0.002 0.00 0.00 0.0000.31 0.15 0.016 Stearic 18:0 0.007 0.48 0.09 0.020 1.10 0.54 0.056 Oleic18:1ω9 0.067 5.50 0.99 0.231 4.83 2.36 0.247 Oleic 18:1ω7 0.014 1.570.28 0.066 1.24 0.61 0.063 Linoleic 18:2ω6 0.053 4.77 0.86 0.200 3.181.56 0.163 Linoleic 18:2ω4 0.000 0.00 0.00 0.000 0.00 0.00 0.000Gamma-Linolenic 18:3ω6 0.000 0.24 0.04 0.010 0.24 0.12 0.012Alpha-Linolenic (ALA) 18:3ω3 0.005 0.40 0.07 0.017 0.36 0.18 0.019Eicosadienoic 20:2ω6 0.000 0.00 0.00 0.000 0.00 0.00 0.000Eicosatrienoic 20:3ω6 0.000 0.00 0.00 0.000 0.00 0.00 0.000 Arachidonic20:4ω6 0.050 3.42 0.62 0.143 3.27 1.60 0.167 Eicosapentaenoic (EPA)20:5ω3 0.282 19.78 3.58 0.831 19.08 9.33 0.975 Other n/a 0.202 13.862.51 0.582 19.29 9.43 0.986 Total Fatty Acid 1.20 100.00 18.07 4.20100.00 48.89 5.11 Total Omega-3 0.29 20.18 3.65 0.85 19.44 9.51 0.99Total Omega-6 0.10 8.42 1.52 0.35 6.69 3.27 0.34

The data shows that warm Hex/MeOH is more effective at extracting fattyacids and Omega 3 from the biomass than any of the room temperatureextraction techniques. Dry DME is next most effective extraction method(5.11 wt % TFA versus 14.5 wt % TFA for 70/30 Hex/MeOH). Drying and roomtemperature extraction significantly inhibits the extraction of fattyacids.

With respect to the room temperature DME extraction, there was apossibility that the material could be either rehydrated or that thepresence of water could assist in the extraction, perhaps as acting as aco-solvent with the DME. Two additional variants on DME were, thus,executed. This used the same biomass lot as in Table 8. This data ispresented in Table 9. The “Wet DME” method involved a light applicationof water to the spray dried biomass. This amounted to the addition ofless than 10% by weight of water to the dried biomass. The intent was torehydrate the surface of the dried algae cells. This material wasallowed to sit for an hour before processing. During processing, the DMEwas passed through a water bath prior to making contact with thebiomass. This saturated the DME with water, as neat DME at roomtemperature has approximately a 6 wt % solubility for water. The“H₂O/MeOH-DME” method involved saturating the spray dried S14 with a75/25 w/w % mixture of H₂O/MeOH. The biomass and liquid was allowed tosoak overnight. This approach was intended to rehydrate the materialwith a mixture that enable efficient DME extraction. 75/25 w/w %H₂O/MeOH is miscible with DME and, thus, is enabling for a much smalleramount of DME to extract this water mixture and the lipids. The data intable 9 show that Wet DME may have a slight improvement over Dry DME.The extraction yielded 6.96 wt % TFA and 1.27 wt % EPA from the biomasswith Wet DME versus 5.11 wt % TFA and 0.975 wt % EPA for Dry DME. Thiswas inferior to 70/30 Hex/MeOH done at Soxhlet like conditions.

TABLE 9 DME Extraction Variants with Spray Dried S14 70/30 (v/v %)Hex/MeOH Dry DME Wet DME H₂O/MeOH— Dry DME Test NJFL Hex/MeOH, 1112-2005QLTS-8 QLTS-13 QLTS-14 Percent Extract from Dry Biomass 34.7 11.0 40.515.1 FA in FA in FA in FA in FA in Dry FA in Dry FA in Dry FA in DryC#:Dbl. Norm Extract Solid Norm Extract Solid Norm Extract Solid NormExtract Solid Fatty Acid Bond FA % % % FA % % % FA % % % FA % % %Caprylic  8:0 0.19 0.08 0.027 0.45 0.22 0.023 0.34 0.06 0.02 0.31 0.100.015 Capric 10:0 0.12 0.05 0.018 0.89 0.44 0.046 1.65 0.28 0.11 0.310.10 0.015 Lauric 12:0 0.39 0.16 0.056 0.68 0.33 0.035 0.70 0.12 0.050.53 0.17 0.025 Myristic 14:0 4.13 1.73 0.599 5.04 2.46 0.258 4.47 0.770.31 4.79 1.50 0.227 Myristoleic 14:1 0.08 0.03 0.011 0.08 0.04 0.0040.00 0.00 0.00 0.14 0.04 0.007 Pentadecanoic 15:0 0.25 0.10 0.036 0.290.14 0.015 0.34 0.06 0.02 0.38 0.12 0.018 Palmitic 16:0 22.32 9.33 3.23620.84 10.19 1.065 21.76 3.75 1.52 22.81 7.13 1.080 Palmitoleic 16:124.72 10.34 3.585 18.40 8.99 0.940 18.60 3.20 1.30 20.12 6.29 0.952Hexadecadienoic 16:2 0.22 0.09 0.032 0.28 0.14 0.014 0.79 0.14 0.06 0.000.00 0.000 Hexadecatrienoic 16:3 0.11 0.05 0.016 0.14 0.07 0.007 0.000.00 0.00 0.00 0.00 0.000 Heptadecanoic 17:0 0.34 0.14 0.050 0.31 0.150.016 0.44 0.07 0.03 0.30 0.09 0.014 Stearic 18:0 0.79 0.33 0.115 1.100.54 0.056 1.19 0.21 0.08 0.72 0.22 0.034 Oleic 18:1ω9 3.12 1.30 0.4524.83 2.36 0.247 4.96 0.85 0.35 5.29 1.65 0.250 Oleic 18:1ω7 0.42 0.180.061 1.24 0.61 0.063 1.44 0.25 0.10 1.20 0.38 0.057 Linoleic 18:2ω62.93 1.23 0.425 3.18 1.56 0.163 3.48 0.60 0.24 4.38 1.37 0.207 Linoleic18:2ω4 0.00 0.00 0.000 0.00 0.00 0.000 0.00 0.00 0.00 0.00 0.00 0.000Gamma-Linolenic 18:3ω6 0.95 0.40 0.137 0.24 0.12 0.012 0.25 0.04 0.020.20 0.06 0.010 Alpha-Linolenic (ALA) 18:3ω3 0.88 0.37 0.128 0.36 0.180.019 0.53 0.09 0.04 0.42 0.13 0.020 Eicosadienoic 20:2ω6 0.00 0.000.000 0.00 0.00 0.000 0.00 0.00 0.00 0.00 0.00 0.000 Eicosatrienoic20:3ω6 0.33 0.14 0.047 0.00 0.00 0.000 0.00 0.00 0.00 0.00 0.00 0.000Arachidonic 20:4ω6 6.05 2.53 0.878 3.27 1.60 0.167 3.16 0.54 0.22 3.160.99 0.150 Eicosapentaenoic (EPA) 20:5ω3 21.33 8.92 3.092 19.08 9.330.975 18.24 3.14 1.27 19.01 5.94 0.900 Other n/a 10.34 4.32 1.499 19.299.43 0.986 17.48 3.01 1.22 15.93 4.98 0.754 Total Fatty Acid 100.0041.81 14.50 100.00 48.89 5.11 99.81 17.18 6.96 100.00 31.26 4.73 TotalOmega-3 22.21 9.29 3.22 19.44 9.51 0.99 18.77 3.23 1.31 19.43 6.07 0.92Total Omega-6 10.26 4.29 1.49 6.69 3.27 0.34 6.89 1.19 0.48 7.74 2.420.37

Table 10 compares drying technique with spray dried S14 versus freezedried S14. Biomass was extracted with Dry DME with the method associatedwith the data in Tables 8 and 9. The biomass was harvested in northernIsrael. The biomass was taken from two different harvest days; however,the harvest is reflective of normal culture during this season of theyear. The freeze dried biomass had an exceptionally low moisture contentof 2.1 wt %. The TFA on a biomass basis was 4.73 wt % for spray driedversus 3.22 wt % for freeze dried. The lower extraction yield could be aresult of the exceptionally low moisture content. For purposes ofmaximizing the recovery of EPA from the biomass, freeze dried materialappears to be at a disadvantage to spray drying.

Table 11 contains the most direct comparison of S14 extraction in a dryversus wet state. The S14 was grown in New Mexico and harvested on asingle day. The paste was 30.8 wt % solid. The wet biomass was combinedwith diatomaceous earth (DE) in a 1:1 w:w ratio prior to being loadedinto the DME extraction. The dry biomass was created by room temperaturefreeze drying. The wet paste was subject to a vacuum of 50 mbar for 48hour period. This results in an 89.2 wt % solid. This material, too, wascombined with DE in 1:1 w:w prior to extraction. In both cases, dry DMEwas used to extract the material. As shown in Table 11, the wet pasteyielded 16.8 wt % TFA and 3.32 wt % EPA from the biomass versus 6.12 wt% TFA and 1.10 wt % EPA with dried biomass. The same biomass yieldedmore than 2.5 times the lipid and over 3 times the EPA in the wet stateversus the dry state. The CAE was also more highly concentrated from thewet paste versus the dried biomass as reflected in the TFA of 50 wt %and 38 wt % and the EPA content of 9.87 wt % and 6.90 wt %. Thus, thereis a dramatic advantage to extracting biomass in the wet state versusthe dry state. Without being bound by a particular theory, presence ofintracellular and extracellular water is enabling for maintaining thecell membrane porosity to the DME solvent, better enabling lipidextraction.

On the basis of data shown in Table 11 for S14 and in Table 7 for S12,we have surprisingly found that the extract from wet Nanno Paste leadsto between 1.5 and 3.5 times more fatty acid recovery from the biomassversus the extraction of the same biomass after drying. In spite of noparticular effort to disrupt the cell membrane via mechanical, thermal,or pH disruption, the wet paste has a higher extraction yield than thesame biomass after drying.

TABLE 10 DME Extraction Comparing Spray Dried S14 and Freeze Dried S14Dry DME- Spray Dried S14 Dry DME- Freeze Dried S14 Test QLTS-8 QLTS-9Percent Extract from Dry Biomass 11.0 7.3 FA in FA in FA in Dry FA inDry C#:Dbl. Norm. Extract Solid Norm. Extract Solid Fatty Acid Bond FA %% % FA % % % Caprylic  8:0 0.45 0.22 0.023 0.47 0.20 0.015 Capric 10:00.89 0.44 0.046 1.11 0.49 0.036 Lauric 12:0 0.68 0.33 0.035 0.52 0.230.017 Myristic 14:0 5.04 2.46 0.258 3.95 1.74 0.127 Myristoleic 14:10.08 0.04 0.004 0.07 0.03 0.002 Pentadecanoic 15:0 0.29 0.14 0.015 0.200.09 0.006 Palmitic 16:0 20.84 10.19 1.065 15.73 6.92 0.506 Palmitoleic16:1 18.40 8.99 0.940 15.05 6.62 0.485 Hexadecadienoic 16:2 0.28 0.140.014 0.42 0.19 0.014 Hexadecatrienoic 16:3 0.14 0.07 0.007 0.00 0.000.000 Heptadecanoic 17:0 0.31 0.15 0.016 0.31 0.14 0.010 Stearic 18:01.10 0.54 0.056 0.68 0.30 0.022 Oleic 18:1ω9 4.83 2.36 0.247 2.92 1.280.094 Oleic 18:1ω7 1.24 0.61 0.063 0.85 0.37 0.027 Linoleic 18:2ω6 3.181.56 0.163 2.48 1.09 0.080 Linoleic 18:2ω4 0.00 0.00 0.000 0.00 0.000.000 Gamma-Linolenic 18:3ω6 0.24 0.12 0.012 0.28 0.12 0.009Alpha-Linolenic (ALA) 18:3ω3 0.36 0.18 0.019 1.27 0.56 0.041Eicosadienoic 20:2ω6 0.00 0.00 0.000 0.08 0.04 0.003 Eicosatrienoic20:3ω6 0.00 0.00 0.000 0.25 0.11 0.008 Arachidonic 20:4ω6 3.27 1.600.167 4.46 1.96 0.144 Eicosapentaenoic (EPA) 20:5ω3 19.08 9.33 0.97525.77 11.34 0.830 Other n/a 19.29 9.43 0.986 23.12 10.17 0.745 TotalFatty Acid 100.00 48.89 5.11 100.00 43.99 3.22 Total Omega-3 19.44 9.510.99 27.04 11.90 0.87 Total Omega-6 6.69 3.27 0.34 7.56 3.33 0.24

TABLE 11 DME Extraction Comparing Dry versus Wet S14 Dry S14- Dry DMES14 Paste- Dry DME Test QLTS-1 QLTS-2 Percent Extract from Dry Biomass16.0 33.7 FA in FA in FA in Dry FA in Dry C#:Dbl. Norm. Extract SolidNorm. Extract Solid Fatty Acid Bond FA % % % FA % % % Caprylic  8:0 0.170.07 0.01 0.15 0.07 0.02 Capric 10:0 1.17 0.45 0.07 1.52 0.76 0.26Lauric 12:0 0.45 0.17 0.03 0.41 0.21 0.07 Myristic 14:0 4.83 1.85 0.304.54 2.27 0.76 Myristoleic 14:1 0.13 0.05 0.01 0.09 0.05 0.02Pentadecanoic 15:0 0.42 0.16 0.03 0.36 0.18 0.06 Palmitic 16:0 22.528.62 1.38 23.48 11.75 3.95 Palmitoleic 16:1 24.37 9.33 1.49 24.32 12.174.09 Hexadecadienoic 16:2 0.45 0.17 0.03 0.25 0.13 0.04 Hexadecatrienoic16:3 0.00 0.00 0.00 0.00 0.00 0.00 Heptadecanoic 17:0 0.41 0.16 0.020.31 0.15 0.05 Stearic 18:0 1.20 0.46 0.07 1.04 0.52 0.18 Oleic 18:1ω92.63 1.01 0.16 2.99 1.50 0.50 Oleic 18:1ω7 0.44 0.17 0.03 0.45 0.23 0.08Linoleic 18:2ω6 1.99 0.76 0.12 2.40 1.20 0.40 Linoleic 18:2ω4 0.00 0.000.00 0.00 0.00 0.00 Gamma-Linolenic 18:3ω6 0.27 0.10 0.02 0.48 0.24 0.08Alpha-Linolenic (ALA) 18:3ω3 0.97 0.37 0.06 0.84 0.42 0.14 Eicosadienoic20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00 Eicosatrienoic 20:3ω6 0.28 0.110.02 0.31 0.15 0.05 Arachidonic 20:4ω6 4.50 1.72 0.28 4.79 2.40 0.81Eicosapentaenoic (EPA) 20:5ω3 18.02 6.90 1.10 19.73 9.87 3.32 Other n/a14.77 5.65 0.90 11.54 5.78 1.94 Total Fatty Acid 100.00 38.27 6.12100.00 50.04 16.84 Total Omega-3 18.99 7.27 1.16 20.57 10.29 3.46 TotalOmega-6 7.04 2.69 0.43 7.98 3.99 1.34

The same S14 biomass lot employed to generate the data in Table 11 wasextracted by a number of different solvent systems in the wet state. Asshown in Table 12, this included DME, 50/50 w/w % acetone/EtOH, 90/10w/w % Acetone/MeOH, and 95/5 v/v % (190 proof) denatured EtOH. The TFAyield based on biomass for the different techniques was 16.84, 13.96,8.95, and 16.12 wt %. We have observed variance in extractionperformance on the order of 15%. Thus, the DME, 50/50 w/w % and 95/5 v/v% EtOH all give about the same TFA yield. From the standpoint of EPAextracted from the biomass, the trend is similar with 3.32, 2.79, 1.71,and 3.41 wt %. In terms of other components extracted with fatty acid,the EPA content in the extract was 9.9, 6.6, 6.4, and 7.1 wt %. The DMEand 95/5 v/v % EtOH provided the best results.

In addition to the fatty acid profile, the polar lipids and otherphytonutrients was determined by Spectral Service GmbH (Cologne,Germany) using a combination of ³¹P NMR (31P NMR), ¹H-NMR (1H NMR), and¹³C-NMR (13C NMR). All spectra were acquired using a Bruker Avance III600 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) with automatedsample changer and QNP cryo probe. Qualitative 31P NMR was according tomethod SAA MET002 02. 1H NMR/13C NMR analysis was according to methodSAA MET001-02. Bruker TopSpin was used for acquisition and dataprocessing. For 31P NMR, the internal standard was triphenyl phosphate(TPP) (Alrich Chemia AG, Buchs, Czech Republic). For the 1H NMR and 13CNMR, the internal standards were TPP and D Sorbitol (C6H14O6, SigmaAldrich, Steinheim, Germany). 31P NMR was used to quantify thephospholipid distribution in the samples. 1H NMR was used to quantifythe digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglyercol(MGDG), cholesterol, chlorophyll. DGDG and MGDG are glycolipids (GL).Polar lipids (PoL) are comprised of phospholipids and glycolipids. TheCholesterol was a marker, in general, for phytosterols and is,hereafter, referred to as phytosterols, total sterols, or sterols. 13CNMR was used to quantify mannitol and glycerol. Mannitol is a linear C6carbohydrate. This constituent has not been previously identified inNannochloropsis oculata extract.

TABLE 12 Wet S14 Paste Extracted with DME and Various Solvent Mixtures50/50 w/w % 90/10 w/w % 95/5 v/v % DME Acetone/EtOH Acetone/MeOH EtOHTest QLTS-2 QLTS-6 QLTS-3 QLTS-7 Percent Extract from Dry Biomass 33.742.2 26.6 48.4 FA in FA in FA in FA in FA in Dry FA in Dry FA in Dry FAin Dry C#:Dbl. Norm. Extract Solid Norm. Extract Solid Norm. ExtractSolid Norm. Extract Solid Fatty Acid Bond FA % % % FA % % % FA % % % FA% % % Caprylic  8:0 0.15 0.07 0.025 0.16 0.05 0.022 0.10 0.03 0.009 0.170.06 0.028 Capric 10:0 1.52 0.76 0.256 0.91 0.30 0.128 1.72 0.58 0.1540.98 0.33 0.158 Lauric 12:0 0.41 0.21 0.070 0.44 0.14 0.061 0.45 0.150.041 0.44 0.15 0.071 Myristic 14:0 4.54 2.27 0.764 4.62 1.53 0.645 4.681.57 0.418 4.50 1.50 0.726 Myristoleic 14:1 0.09 0.05 0.016 0.15 0.050.021 0.10 0.03 0.009 0.10 0.03 0.016 Pentadecanoic 15:0 0.36 0.18 0.0610.34 0.11 0.047 0.33 0.11 0.029 0.38 0.13 0.061 Palmitic 16:0 23.4811.75 3.953 22.59 7.47 3.155 23.72 7.98 2.122 23.00 7.66 3.707Palmitoleic 16:1 24.32 12.17 4.095 24.69 8.17 3.448 25.59 8.62 2.28924.99 8.32 4.029 Hexadecadienoic 16:2 0.25 0.13 0.043 0.22 0.07 0.0310.11 0.04 0.010 0.24 0.08 0.038 Hexadecatrienoic 16:3 0.00 0.00 0.0000.13 0.04 0.018 0.00 0.00 0.000 0.01 0.00 0.002 Heptadecanoic 17:0 0.310.15 0.052 0.37 0.12 0.051 0.39 0.13 0.034 0.41 0.14 0.066 Stearic 18:01.04 0.52 0.175 0.69 0.23 0.097 1.00 0.34 0.090 0.66 0.22 0.106 Oleic18:1ω9 2.99 1.50 0.503 3.09 1.02 0.431 2.92 0.98 0.262 3.28 1.09 0.528Oleic 18:1ω7 0.45 0.23 0.076 0.52 0.17 0.072 0.58 0.19 0.052 0.47 0.160.075 Linoleic 18:2ω6 2.40 1.20 0.405 2.73 0.90 0.381 2.43 0.82 0.2172.74 0.91 0.441 Linoleic 18:2ω4 0.00 0.00 0.000 0.05 0.02 0.006 0.000.00 0.000 0.00 0.00 0.000 Gamma-Linolenic 18:3ω6 0.48 0.24 0.081 0.610.20 0.085 0.47 0.16 0.042 0.70 0.23 0.113 Alpha-Linolenic (ALA) 18:3ω30.84 0.42 0.142 1.06 0.35 0.148 0.91 0.30 0.081 0.99 0.33 0.160Eicosadienoic 20:2ω6 0.17 0.09 0.029 0.13 0.04 0.018 0.10 0.03 0.0090.07 0.02 0.012 Eicosatrienoic 20:3ω6 0.31 0.15 0.052 0.36 0.12 0.0510.34 0.12 0.031 0.27 0.09 0.044 Arachidonic 20:4ω6 4.79 2.40 0.807 4.821.60 0.674 4.87 1.64 0.436 5.39 1.79 0.869 Eicosapentaenoic (EPA) 20:5ω319.73 9.87 3.322 19.96 6.60 2.787 19.08 6.43 1.707 21.18 7.05 3.414Other n/a 11.37 5.69 1.915 11.36 3.76 1.587 10.12 3.41 0.905 9.03 3.011.455 Total Fatty Acid 100.00 50.04 16.84 100.00 33.07 13.96 100.0033.67 8.95 100.00 33.30 16.12 Total Omega-3 20.57 10.29 3.46 21.02 6.952.93 19.99 6.73 1.79 22.17 7.38 3.57 Total Omega-6 8.15 4.08 1.37 8.662.86 1.21 8.21 2.76 0.73 9.17 3.05 1.48

TABLE 13 Polar Lipids of S12 and S14 Compared with Krill Oil FishRefined Krill Nannochloropsis Oculata Oil EPA Oil Lipid Class Wet DryWet Dry Ethyl Plus NOW Component Name Code S12 S12 S14 S14 Ester EPA NKOPhospholipid Phosphatidylcholine PC 3.09 2.33 8.55 4.46 0.00 0.00 27.73Lyso-Phosphatidylcholine 1-LPC 0.00 0.00 0.00 0.00 0.00 0.00 0.39Lyso-Phosphatidylcholine 2-LPC 0.00 0.00 0.41 0.36 0.00 0.00 3.45Phosphatidylinositol PI 1.35 0.00 1.95 1.26 0.00 0.00 0.27Lyso-Phosphatidylinositol LPI 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidylserine PS-Na 0.00 0.00 0.00 0.00 0.00 0.00 0.00Lyso-Phosphatidylserine LPS 0.00 0.00 0.00 0.00 0.00 0.00 0.00Sphingomyelin SPH 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidylethanolamine PE 0.66 0.00 2.28 0.50 0.00 0.00 2.72Lyso-Phosphatidylethanolamine LPE 0.00 0.00 0.00 0.00 0.00 0.00 0.40N-Acyl-Phosphatidylethanolamine APE 0.00 0.00 0.00 0.00 0.00 0.00 1.26Phosphatidylglycerol PG 2.63 2.30 4.91 2.31 0.00 0.00 0.00Diphosphatidylglycerol DPG 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidic Acid PA 0.00 0.00 0.00 0.00 0.00 0.00 0.00Lyso-Phosphatidic Acid LPA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Other 1.111.77 1.25 1.08 0.00 0.00 0.28 Glycolipid Digalactosyldiacylglycerol DGDG17.25 7.61 18.35 16.36 0.00 0.00 0.00 Monogalactosyldiacylglycerol MGDG5.40 2.61 6.18 4.28 0.00 0.00 0.00 Total Phospholipids (PL) (wt %) 8.846.39 19.34 9.96 0.00 0.00 36.50 Total Glycolipids (GL) (wt %) 22.6510.22 24.53 20.64 0.00 0.00 0.00 Total PoL (PL + GL) (wt %) 31.49 16.6143.86 30.60 0.00 0.00 36.50

Table 13 shows the polar lipid composition of semi-refined DME extractsof S12 and S14 biomass versus krill oil. The semi-refined materialinvolves a water partition that removes approximately half of the watersoluble, non-lipid constituents in the crude algae extract (CAE). Byfar, the largest difference between krill oil and Nannochloropsisoculata extract is that krill oil contains no GL. Krill lack thebiosynthetic pathways to produce GLs. Furthermore, N. oculata producesless phospholipid than krill oil. N. oculata produces more GL than PL,in a range between 20% and 300% more GL than PL. The table shows theeffect of spray drying versus wet paste. Wet paste enables between 50and 100% more polar lipids to be extracted as reflected by the Total PoLcontents of Wet S12 and Dry S12 of 31.5 and 16.6 and of Wet S14 and DryS14 of 43.9 and 30.6 wt %. All S12 and S14 extract containsphosphatidylcholine (PC), phosphatidylglycerol (PG), and otherphospholipid components. Except for Dry S12, S12 and S14 also havephosphatidylinositol (PI) and phosphatidylethanolamine (PE). S12 and S14is devoid of 1 Lyso-Phosphatidylcholine (1 LPC),Lyso-Phosphatidylethanolamine (LPE), and N Acyl Phosphatidylethanolamine(APE) that are found in krill oil.

Table 14 shows the polar lipid content of dried versus wet S14 algae.This is the polar and phytonutrient analysis of QLTS 1 and QLTS 2. Thisextract is Crude Algae Extract (CAE) per the earlier definition. The FAPfrom extractions is reported in Table 11. For exactly the same lot ofbiomass, extraction via DME in the wet state yielded 4.34 wt % PL and5.50 wt % GL on a biomass basis versus extraction via DME In the drystate which yielded 0.61 wt % PL and 0.81 wt % GL on a biomass basis.Wet state extract was more than 7 times more effective in extracting PLand over 6 times more effective in extracting GL. Phytosterols andchlorophyll are extracted 2 times more effectively in the wet stateversus the dry state. The CAE from wet extraction has 12.89 wt % PL and13.35 wt % GL. For dry extraction, the PL is 3.81 wt % and 5.06 wt %.CAE is over three times more concentrated in PL and more than 2.5 timesmore concentrated in GL. From this result, we conclude that there is asignificant advantage of wet extraction versus dry extraction.

TABLE 14 Polar Lipids and Phytonutrients from Dry and Wet S14 Extractedwith DME Dry S14- Dry DME S14 Paste- Dry DME Test QLTS-1 QLTS-2 PercentExtract from Dry Biomass 16.0 33.7 Class Extract Biomass Extract BiomassComponent Name Code Basis % Basis % Basis % Basis % PhospholipidPhosphatidylcholine PC 1.43 0.23 5.70 1.92 Lyso-Phosphatidylcholine1-LPC 0.00 0.00 0.00 0.00 Lyso-Phosphatidylcholine 2-LPC 0.00 0.00 0.270.09 Phosphatidylinositol PI 0.54 0.09 1.30 0.44Lyso-Phosphatidylinositol LPI 0.00 0.00 0.00 0.00 PhosphatidylserinePS-Na 0.00 0.00 0.00 0.00 Lyso-Phosphatidylserine LPS 0.00 0.00 0.000.00 Sphingomyelin SPH 0.00 0.00 0.00 0.00 Phosphatidylethanolamine PE0.53 0.08 1.52 0.51 Lyso-Phosphatidylethanolamine LPE 0.00 0.00 0.000.00 N-Acyl-Phosphatidylethanolamine APE 0.00 0.00 0.00 0.00Phosphatidylglycerol PG 0.96 0.15 3.27 1.10 Diphosphatidylglycerol DPG0.00 0.00 0.00 0.00 Phosphatidic Acid PA 0.00 0.00 0.00 0.00Lyso-Phosphatidic Acid LPA 0.00 0.00 0.00 0.00 Other 0.35 0.06 0.83 0.28Glycolipid Digalactosyldiacylglycerol DGDG 3.91 0.63 12.23 4.12Monogalactosyldiacylglycerol MGDG 1.15 0.18 4.12 1.39 PhytonutrientsPhytosterols 2.23 0.36 2.43 0.82 Chlorophyll 6.73 1.08 6.54 2.20 TotalPhospholipids (PL) (wt %) 3.81 0.61 12.89 4.34 Total Glycolipids (GL)(wt %) 5.06 0.81 16.35 5.50 Total PoL (PL + GL) (wt %) 8.87 1.42 29.249.84

Table 15 compares different solvent systems in the extraction yield ofpolar lipids and phytonutrients from S14 algae paste. The FAP fromextractions is reported in Table 12. The solvent systems were DME, 50/50w/w % acetone/EtOH (Ace/EtOH), 90/10 w/w % Acetone/MeOH (Ace/MeOH), and95/5 v/v % (190 proof) denatured EtOH (190 proof EtOH). On a biomassbasis, the PL content was 4.34, 3.83, 1.53, and 4.79 and the GL contentwas 5.50, 3.88, 1.62, and 4.76 for DME, Ace/EtOH, Ace/MeOH, and 190proof EtOH. The extraction yields were approximately the same for DMEand 190 proof EtOH. Ace/EtOH could be lower due to sample to samplevariance. In terms of the CAE, DME was the best at 29.24 wt % Total PoL,while Ace/EtOH and 190 proof EtOH were 18.26 and 19.73 wt %,respectively. We know from later work, that the liquid solvents have agreater amount of water-soluble non-lipid components than the DMEextract. This water-soluble component is removed during the conversionof CAE to CAO. Note that all the liquid solvents on wet paste stillproduce better yield of PoL from the biomass and higher concentrationsof PoL in the extract than the dry biomass extracted with DME (See,Table 14).

Table 16 shows the polar lipid content of dried versus wet S12 algae.This is the polar and phytonutrient analysis of QLTS 18 and QLTS 17. TheFAP from these extractions is reported in Table 7. For biomass harvestedin the same time period and for DME extraction results on a biomassbasis, the wet state S12 yielded 3.53 wt % PL and 9.06 wt % GL on abiomass basis versus the dry state yielding 2.13 wt % PL and 3.41 wt %GL on a biomass basis. Wet state extract is more than 1.5 times moreeffective in extracting PL and more than 2.5 times more effective inextracting GL. The CAE from wet extraction has 5.89 wt % PL and 15.10 wt% GL. For dry extraction, the PL is 4.26 wt % and 6.81 wt %. CAE is over33% more concentrated in PL and nearly 2.5 more concentrated in GL. Thisresult further reinforces that there is a significant advantage of wetextraction over dry extraction.

TABLE 15 Polar Lipids and Phytonutrients from Wet S14 Paste Extractedwith DME and Other Solvent Systems 50/50 w/w % 90/10 w/w % 95/5 v/v %DME Ace/EtOH Ace/MeOH EtOH Test QLTS-2 QLTS-6 QLTS-3 QLTS-7 PercentExtract from Dry Biomass 33.7 42.2 26.6 48.4 Class Extract BiomassExtract Biomass Extract Biomass Extract Biomass Component Name CodeBasis % Basis % Basis % Basis % Basis % Basis % Basis % Basis %Phospholipid Phosphatidylcholine PC 5.70 1.92 4.16 1.76 2.93 0.78 3.811.84 Lyso-Phosphatidylcholine 1-LPC 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 Lyso-Phosphatidylcholine 2-LPC 0.27 0.09 0.19 0.08 0.13 0.03 0.290.14 Phosphatidylinositol PI 1.30 0.44 0.60 0.25 0.28 0.07 0.91 0.44Lyso-Phosphatidylinositol LPI 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidylserine PS-Na 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Lyso-Phosphatidylserine LPS 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Sphingomyelin SPH 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidylethanolamine PE 1.52 0.51 1.12 0.47 0.47 0.13 1.26 0.61Lyso-Phosphatidylethanolamine LPE 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 N-Acyl-Phosphatidylethanolamine APE 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 Phosphatidylglycerol PG 3.27 1.10 2.56 1.08 1.65 0.44 2.841.38 Diphosphatidylglycerol DPG 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Phosphatidic Acid PA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Lyso-Phosphatidic Acid LPA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Other0.83 0.28 0.42 0.18 0.29 0.08 0.78 0.38 GlycolipidDigalactosyldiacylglycerol DGDG 12.23 4.12 6.37 2.69 4.19 1.11 6.71 3.25Monogalactosyldiacylglycerol MGDG 4.12 1.39 2.83 1.19 1.90 0.51 3.121.51 Phytonutrients Phytosterols 2.43 0.82 1.55 0.65 2.08 0.55 1.42 0.69Chlorophyll 6.54 2.20 4.78 2.02 3.65 0.97 4.61 2.23 Total Phospholipids(PL) (wt %) 12.89 4.34 9.06 3.83 5.75 1.53 9.90 4.79 Total Glycolipids(GL) (wt %) 16.35 5.50 9.20 3.88 6.09 1.62 9.83 4.76 Total PoL (PL + GL)(wt %) 29.24 9.84 18.26 7.71 11.85 3.15 19.73 9.55

TABLE 16 Polar Lipids and Phytonutrients from DME Extracted Dry and WetS12 Spray Dried S12- Wet DME Wet S12 Slurry- Dry DME Test QLTS-18QLTS-17 Percent Extract from Dry Biomass 46.0 60.0 Class Extract BiomassExtract Biomass Component Name Code Basis % Basis % Basis % Basis %Phospholipid Phosphatidylcholine PC 1.55 0.78 2.06 1.24Lyso-Phosphatidylcholine 1-LPC 0.00 0.00 0.00 0.00Lyso-Phosphatidylcholine 2-LPC 0.00 0.00 0.00 0.00 PhosphatidylinositolPI 0.00 0.00 0.90 0.54 Lyso-Phosphatidylinositol LPI 0.00 0.00 0.00 0.00Phosphatidylserine PS-Na 0.00 0.00 0.00 0.00 Lyso-Phosphatidylserine LPS0.00 0.00 0.00 0.00 Sphingomyelin SPH 0.00 0.00 0.00 0.00Phosphatidylethanolamine PE 0.00 0.00 0.44 0.26Lyso-Phosphatidylethanolamine LPE 0.00 0.00 0.00 0.00N-Acyl-Phosphatidylethanolamine APE 0.00 0.00 0.00 0.00Phosphatidylglycerol PG 1.53 0.77 1.75 1.05 Diphosphatidylglycerol DPG0.00 0.00 0.00 0.00 Phosphatidic Acid PA 0.00 0.00 0.00 0.00Lyso-Phosphatidic Acid LPA 0.00 0.00 0.00 0.00 Other 1.18 0.59 0.74 0.44Glycolipid Digalactosyldiacylglycerol DGDG 5.07 2.54 11.50 6.90Monogalactosyldiacylglycerol MGDG 1.74 0.87 3.60 2.16 PhytonutrientsPhytosterols 0.57 0.29 1.20 0.72 Chlorophyll 4.51 2.26 12.00 7.20 TotalPhospholipids (PL) (wt %) 4.26 2.13 5.89 3.53 Total Glycolipids (GL) (wt%) 6.81 3.41 15.10 9.06 Total PoL (PL + GL) (wt %) 11.07 5.54 20.9912.59

In addition to the greater yield, wet extraction of S12 has anotheradvantage in that the distribution of phospholipids differs from thatfrom dry S12. Note that in QLTS-17, the phospholipid distributionincludes Phosphatidylinositol (PI) and Phosphatidylethanolamine (PE). PIand PE contribute 0.90 and 0.44 wt %, respectively, of the total 5.89 wt% PL in the CAE. PI and PE represent 15.2% and 9.0% by mass of the totalPL, nearly 25% of the mixture. The difference in the PL distribution isalmost completely accounted for by these two missing constituents. WithS12 Nannochloropsis oculata, it appears that the presence ofintracellular water is essential for the extraction of these two PLconstituents.

A CAE comprised of NL, PL, GL, chlorophyll, sterols, carotenoids,manitol, and glycerol results from these different drying approaches,pretreatment methods, and solvent extraction methods on S12 and S14. PLand GL together comprise the polar lipids. The phospholipids in S12 andS14 are PC (Phosphatidylcholine), PI (Phosphatidylinositol), PE(Phosphatidylethanolamine), PG (Phosphatidylglycerol), and othernon-specific phospholipids. Based on Table 14, there are higherproportions of “other” phospholipids in S12 and S14 oil than in krilloil. This implies that there are a greater number of unique PL compoundsin S12 and S14 oil than in krill oil. The following phospholipids arenotably absent in S12 and S14 N. Oculata oil: LPI(Lyso-Phosphatidylinositol), PS (Phosphatidylserine), LPS (LysoPhosphatidylserine), SPH (Sphingomyelin), LPE (LysoPhosphatidylethanolamine), APE (N Acyl Phosphatidylethanolamine), PA(Phosphatidic Acid), and LPA (Lyso Phosphatidic Acid). The glycolipidsare DGDG (Digalactosyldiacylglycerol) and MGDG(Monogalactosyldiacylglycerol). There is zero GL in krill oil.

As shown in FIG. 6, the CAE contains a significant proportion of MONL.MONL is material not otherwise accounted for in the TFA, phospholipids,glycolipids, and phytonutrients. MONL is believed to be water-solublecarbohydrates and proteins; however, the composition of this fraction iscurrently unexplored. CAE can be converted to CAO through waterpartitioning. MONL refinement may include a partition of thewater-soluble components comprising excess water to CAE, bringing thewater and CAE into intimate contact with a high shear mixer, andseparation of the water and organic phase via either settling orcentrifugation. The organic phase is the CAO and is a lipid rich mixtureof polar lipids (PoL) and neutral lipids (NL). Alternatively, CAE can beextracted in series with a solvent more suitable for neutral lipids,such as hexane, chloroform, cyclohexane, methylene chloride, orcombinations thereof, followed by further extraction by a solventsuitable for PoL, such as acetone, methanol, ethanol or combinationthereof. In the first step, the solution partitions and the NL richupper phase is collected. A NL rich extract is recovered by evaporatingthe solvent. The bottom phase, now rich in both PoL and MONL, isextracted with a PoL suitable solvent system. After extraction, the PoLrich extract is recovered by evaporating the solvent. CAO results whenthe NL rich and PoL rich extracts are combined. The conversion from CAEto CAO results in between 30 and 50% reduction in mass. CAO is one ofthe constituents of the EPA standardized EPA/Polar Lipid blend.

The composition of CAE, CAO, and the distribution of FA among thedifferent lipid classes for S12 Nannochloropsis are shown in FIGS. 6, 7,and 8, respectively. CAE of S12 contains between 35 and 45 wt % MONL.This MONL is substantially reduced in CAO. In CAO, MONL is between 4 and10 wt % of the CAO. Fatty acid is distributed between NL other than FFA,FFA, PL, and GL. In S12, the FA is almost equally divided between NL andPoL. The TG/DG NLs are between 35 and 48% of the FA, and the FFA NL isbetween 3 and 12%, with 6% being typical. Exposure to elevatedtemperature in harvesting or in extraction can result in the conversionof FA with GL, PL, or TG/DG to FFA. This is a hydrolysis process. Careis taken to minimize elevated temperature and to reduce the time at anytemperature above ambient. Of the PoL, the GL represents about ⅗ of theFA in PoL (29%), and the PL represents about ⅖ of the FA in the PoL(21%). These numbers could fluctuate by ±10% from their nominal valuesbased on growth media and environmental conditions.

The composition of CAE, CAO, and the distribution of FA among thedifferent lipid classes for S14 Nannochloropsis are shown in FIGS. 9,10, and 11, respectively. CAE of S14 contains between 40 and 60 wt %MONL. This MONL is substantially reduced in CAO. In CAO, MONL is between5 and 15 wt % of the CAO. Fatty acid is distributed between NL otherthan FFA, FFA, PL, and GL. In S14, the FA is about 40% PoL and about 60%NL. The TG/DG NLs are between 40 and 50% of the FA, and the FFA NL isbetween 10 and 25%, with 17% being typical. As with S12, exposure toelevated temperature in harvesting or in extraction can result in theconversion of FA with GL, PL, or TG/DG to FFA. Of the PoL, the GLrepresents about half of the FA in PoL (17%), and the PL representsabout half of the FA in the PoL (20%). These numbers could fluctuate by±10% from their nominal values based on growth media and environmentalconditions.

As shown in FIG. 4, CAO can be split into a NL-rich and zero PoL mixturewith high pressure/high temperature (HP/HT) supercritical carbon dioxide(SCCO2). We have found that SCCO2 extracts neutral lipids completely andessentially zero polar lipids in either the form of phospholipids orglycolipids. SCCO2 in the range from 100 to 1000 bar and temperaturesbetween 35 and 110° C. has a high distribution coefficient for neutrallipids and an essentially zero distribution coefficient for polarlipids. Typical values would, at a minimum, be 340 bar and 40° C. up to700 bar and 110° C. In varying embodiments, pressure and temperatureranges are between 350 bar/60° C. and 690 bar/90° C. At 350 bar and 60C., the density of SCCO2 is 0.863 g/mL. At 700 bar/100° C., SCCO2 has adensity of 0.9 g/mL. Process conditions in the pressure range between340 bar and 700 bar that yield a density of 0.83 to 0.9 g/mL aresuitable. High P/T SCCO2 produces a NL fraction with zero PoL. Itextracts a proportion of the chlorophyll and almost all the sterols fromthe CAO. The NL fraction is comprised of free fatty acids (FFA),triglycerides (TG), diglycerides (DG), chlorophyll, and sterols. Theresidual material from high P/T SCCO2 extraction is concentrated polarlipids (Conc PoL), including phosopholipids and glycolipids. The ConcPoL is the second component in the EPA-standardized blend. This streamand the COA provide all the polar lipids for the EPA standardizedEPA/Polar Lipid blend.

As an alternative to the process in FIG. 4, CAE can be split into an NLrich fraction and PoL rich fraction using HP/HT SCCO2 followed byextraction with dimethyl ether (DME). An example of wet paste process byacetone/ethanol to produce CAE, the resultant HT/HP SCCO2 fraction, andDME fraction are presented in Table 17A, 17B, and 17C. Table 17A showsthe fatty acid profile. The most notable characteristics is therelatively low TFA in the CAE. This is due to the presence of MONL.Table 17B shows the polar lipid and phytonutrient composition for thesame samples. The most critical observation from Table 17B is that HP/HTSCCO2 does not extract any polar lipids (PoL). Neither PL nor GL aresoluble in HP/HT SCCO2. SCCO2 extracts a large fraction of the NL. Table17C shows the distribution of FA in the NL, PL, and GL classes and thefraction of NL, PL, and GL in the overall sample. In the CAE, the NL isnearly 75% of the FA and the balance of FA is split almost equallybetween PL and GL. In the HP/HT SCCO2 fraction, the NL is concentratedand there is zero PL and zero GL. The NL goes from being 23.5 wt % ofthe CAE to 69.3 wt % in the NL concentrate (HP/HT SCCO2). The MONL isalmost completely removed in the NL concentrate. Finally, the DME or PoLconcentrate fraction contains virtually all PoL from the CAE. The FAdistribution in the PoL concentrate contains 40.4% NL, 28.0% PL, 31.6%GL. In the total DME sample, total lipids are 62.4 wt % comprised of19.3 wt % NL, 18.9 wt % PL, and 24.3 wt % GL.

TABLE 17A Fatty Acid Composition of S14 CAE, HP/HT SCCO2 Extract and DMEExtract Control High P/T DME after Sample SCCO2 SCCO2 Test QLTS-B-4QLTS-B-5 QLTS-B-5 Extract F1 F2 + F3 Percent of Control Material 24.830.2 FA in FA in FA in C#:Dbl. Norm. Mixture Norm. Fraction Norm.Fraction Fatty Acid Bond FA % % FA % % FA % % Caprylic  8:0 0.21 0.070.20 0.14 0.23 0.11 Capric 10:0 0.12 0.04 0.24 0.16 0.00 0.00 Lauric12:0 0.44 0.14 0.59 0.41 0.26 0.12 Myristic 14:0 4.87 1.54 4.86 3.364.88 2.32 Myristoleic 14:1 0.11 0.03 0.16 0.11 0.05 0.02 Pentadecanoic15:0 0.28 0.09 0.38 0.26 0.16 0.08 Palmitic 16:0 22.70 7.15 25.63 17.7519.12 9.11 Palmitoleic 16:1 25.05 7.89 32.94 22.81 15.58 7.42Hexadecadienoic 16:2 0.21 0.07 0.26 0.18 0.15 0.07 Hexadecatrienoic 16:30.00 0.00 0.17 0.12 0.00 0.00 Heptadecanoic 17:0 0.43 0.13 0.62 0.430.20 0.09 Stearic 18:0 0.79 0.25 1.22 0.85 0.27 0.13 Oleic 18:1ω9 3.060.96 3.57 2.47 2.45 1.17 Oleic 18:1ω7 0.49 0.16 0.58 0.40 0.39 0.19Linoleic 18:2ω6 2.29 0.72 1.82 1.26 2.84 1.35 Linoleic 18:2ω4 0.00 0.000.00 0.00 0.00 0.00 Gamma-Linolenic 18:3ω6 0.63 0.20 0.38 0.26 0.92 0.44Alpha-Linolenic (ALA) 18:3ω3 0.77 0.24 0.80 0.55 0.74 0.35 Arachidic20:0 0.16 0.05 0.37 0.25 0.00 0.00 Eicosatrienoic 20:3ω6 0.40 0.13 0.510.35 0.26 0.13 Arachidonic 20:4ω6 4.73 1.49 3.45 2.39 6.23 2.97Eicosapentaenoic (EPA) 20:5ω3 20.72 6.53 9.92 6.87 33.49 15.96 Other n/a11.53 3.63 11.34 7.85 11.80 5.62 Total Fatty Acid 100.00 31.50 100.0069.25 100.00 47.65 Total Omega-3 21.50 6.77 10.72 7.43 34.23 16.31EPA/Total Omega-3 96.41 92.53 97.85 Total Omega-6 8.20 2.58 6.54 4.5310.25 4.88

TABLE 17B Polar Lipid and Phytonutrient Composition of S14 CAE, HP/HTSCCO2 Extract and DME Extract Control High P/T DME after Sample SCCO2SCCO2 Test QLTS-B-4 QLTS-B-5 QLTS-B-5 Extract F1 F2 + F3 Class RelativeExtract Relative Extract Relative Extract Component Name Code Basis %Basis % Basis % Basis % Basis % Basis % Phospholipid PhosphatidylcholinePC 41.1 2.35 0.00 40.9 7.73 Lyso-Phosphatidylcholine 1-LPC 0.0 0.00 0.000.0 0.00 Lyso-Phosphatidylcholine 2-LPC 3.0 0.17 0.00 1.8 0.34Phosphatidylinositol PI 10.4 0.59 0.00 12.3 2.33Lyso-Phosphatidylinositol LPI 0.0 0.00 0.00 0.0 0.00 PhosphatidylserinePS-Na 0.0 0.00 0.00 0.0 0.00 Lyso-Phosphatidylserine LPS 0.0 0.00 0.000.0 0.00 Sphingomyelin SPH 0.0 0.00 0.00 0.0 0.00Phosphatidylethanolamine PE 8.7 0.50 0.00 9.7 1.83Lyso-Phosphatidylethanolamine LPE 0.0 0.00 0.00 0.0 0.00N-Acyl-Phosphatidylethanolamine APE 0.0 0.00 0.00 0.0 0.00Phosphatidylglycerol PG 25.6 1.47 0.00 25.9 4.90 DiphosphatidylglycerolDPG 0.0 0.00 0.00 0.0 0.00 Phosphatidic Acid PA 0.0 0.00 0.00 0.0 0.00Lyso-Phosphatidic Acid LPA 0.0 0.00 0.00 0.0 0.00 Other 11.2 0.64 0.009.3 1.76 Glycolipid Digalactosyldiacylglycerol DGDG 74.2 4.71 0.00 72.917.68 Monogalactosyldiacylglycerol MGDG 25.8 1.64 0.00 27.1 6.57Phytonutrients Phytosterols 1.45 4.98 0.38 Chlorophyll 4.33 3.44 9.93Total Phospholipids (PL) (wt %) 5.73 0.00 18.90 Total Glycolipids (GL)(wt %) 6.35 0.00 24.24 Total PoL (PL + GL) (wt %) 12.08 0.00 43.14

TABLE 17C Fatty Acid Distribution and NL, PL, GL distribution of S14CAE, HP/HT SCCO2 Extract and DME Extract Control High P/T DME afterSample SCCO2 SCCO2 Test QLTS-B-4 QLTS-B-5 QLTS-B-5 Extract F1 F2 + F3 Wt% % of wt % in % of wt % in % of Compound Class Oil FA Fraction FAFraction FA Fatty Acid as Neutral Lipids (NL) 23.5 74.7 69.3 100.0 19.340.4 Fatty Acid as Phospholipids (PL) 4.0 12.8 0.0 0.0 13.3 28.0 FattyAcid as Glycolipids (GL) 3.9 12.5 0.0 0.0 15.1 31.6 Total Fatty Acid(TFA) 31.5 100.0 69.3 100.0 47.7 100.0 Total NL 23.5 69.3 19.3 Total PL5.7 0.0 18.9 Total GL 6.4 0.0 24.3 Total Lipids = NL + PL + GL 35.6 69.362.4

To create the controlled concentration of EPA in the mixture, the NLfraction must be further concentrated in EPA. As shown in FIG. 1, the NLfraction lipids must be homogenized. This means that any fatty acidsassociated with a glycerol backbone must be separated from thisbackbone. Little concentration of the EPA fatty acid is possible whilethe EPA is conjugated with the glycerol backbone. Suitable methods forFA homogenization include transesterification to form methyl or ethylesters or hydrolysis to create FFA. The preferred method is hydrolysisto create FFA. Transesterification to form methyl or ethyl estersrequires additional process steps and the consumption of methanol orethanol during the transesterification. Hydrolysis can be achievedthrough saponification and acidification or direct pressurized steamhydrolysis. Once the fatty acids are freed of the covalent bond to theglycerol backbone, they can be reorganized according to a combination oftheir molecular weight and degree of unsaturation (i.e., number ofdouble bonds). Many methods can be used to concentrate EPA. For example,urea crystallization can be used to remove the majority of saturated andmonounsaturated FA from the mixture. Furthermore, the fatty acid mixturecan be dissolved in solvent and complexed with silver nitrate or silverfunctionalized silica. This has the net effect of removing the highlypolyunsaturated material from the balance of the mixture. Anotheralternative is to use pressure profiling with SCCO2 to selectivelyremove the lower molecular weight components (i.e., C12-C18) from thehigher molecular weight constituents (i.e. C20).

FIG. 4 shows the combination of hydrolysis and SCCO2 fractionation tocreate a concentrate of EPA FFA. The NL fraction is first hydrolyzed toform FFA. This can be done by a variety of routes that are familiar tolipid chemists. The most common methods are saponification followed byacidification and direct acidification. In terms of product yield,saponification is a useful route because the first step in the reactionirreversibly forms a fatty acid salt. In this case, the neutral lipidmixture is combined with KOH or NaOH in the presence of an excess of thewater. The oxyl bond between the fatty acid and the glycerol backbone isbroken and the respective K or Na salt formed. This reaction iscompleted under reflux at temperature conditions between 50 and 90° C.TG and DG constituents are converted to a salt and free glycerol. Freeglycerol is highly polar. The salt solution is treated with an acid,such as phosphoric, sulfuric, or hydrochloric acid. This removes thesalt's cation and forms the corresponding free fatty acid (FFA). Thesolution partitions into two phases: an organic and aqueous phase. Inthe direct acidification method, the reaction has fewer steps but isreversible. Hence, the yield to FFA may not be as great as thesaponification route. Under acidification, neutral lipid is combinedwith water and strong acid, such as sulfuric, hydrochloric, phosphoric,or formic. Water in excess of stoichiometry, on the order of 6 times, isadded to the neutral lipid. Acid is added to lower the pH toapproximately 2. The mixture is heated under reflux at a temperaturebetween 60 and 100° C. This reaction, while single step, is reversible.An excess of water is required to drive the equilibrium in the directionof FFA.

Once the neutral lipid has been hydrolyzed to form FFA, the EPA fractionwithin this mixture can be further concentrated. Under the previousprocessing step, all the triglycerides and diglycerides have beenconverted to FFA. This is known as high acid oil, a mixture of differentFA compounds that are predominantly in free fatty acid form. While is itknown from the literature that SCCO2 can concentrate Omega-3 from methylesters and, by extension, ethyl esters (Nilsson, et. al., “SupercriticalFluid CO2 Fractionation of Fish Oil Esters” in Advances in SeafoodBiochemistry, 1992), it was not previously known that SCCO2 couldfractionate mixtures of FFA. FFAs are polar moieties. Conventionalthought in SCCO2 solubility is that these compounds would be insolublein SCCO2 and, thus, not be amenable to tunable dissolvingcharacteristics of SCCO2. Surprisingly, we have found that SCCO2 iscapable of fractionating FFAs by molecular weight. Without being boundby any particular theory, the non-polar effect of long carboxylic acidchain from 8 to 20 carbon molecules long overwhelms the polarcharacteristics of the carbonyl group. Thus, in the presence ofisothermal conditions, increasing SCCO2 pressure about 100 bar resultsin increasingly greater solubility for higher molecular weightcarboxylic acids. Using lower pressure SCCO2 at a pressure above 100 barand 40 C can be used to remove the lower molecular weight free fattyacids from the higher molecular weight free fatty acids. This enablesconcentrating the C20 components, including EPA and ARA, while reducingor eliminating the C8, C10, C12, C14, C18 constituents. This enables atleast doubling of the EPA concentration. After concentration, this isthe EPA-Concentrated FFA stream (Conc EPA) and is the third constituentin the mixture to create an EPA-standardized formulation.

Surprisingly, we have found that a high FFA feedstock can befractionated by pressure gradient SCCO2. As an example, a feedstock wasderived from S14 biomass via a hydrolyzing extraction method. Thebiomass was treated with sulfuric acid and heated to 70° C. The mixtureof biomass was then extracted with hexanes. After evaporating thehexanes, a partially hydrolyzed algae oil was recovered. This mixturewas approximately 44.7% FFA with a 80.03 wt % TFA. The composition ofthe feedstock is shown in Table 18. Under 60° C. isothermal conditions,this oil was extracted with a pressure profile, starting at 2500 psi(172 bar) for the first fraction (F1) and increasing 100 psi (6.9 bar)with each subsequent fraction (i.e. 2600 psi (179 bar) for the secondfraction (F2), 2700 psi (186 bar) for the third fraction (F3), etc.).The final pressure was for fraction F12 was 5000 psi (345 bar). Thisfully extracted the feedstock material.

The FFA level and the percentage of the feed for each fraction is shownin Table 19. The FFA levels are shown in the plot in FIG. 12. Both thetable and the figure show high FFA levels in fractions F1 through F6. Infractions F7 through F12, the non-hydrolyzed triglycerides exist. Table20 shows the FA compositions of fractions F1 through F7 and thepercentage of the feedstock mass that was recovered in each of thesefractions. Surprisingly, the lower molecular weight compounds wereconcentrated in fractions F1 through F3. The higher molecular weightcompounds were concentrated in fractions F4 through F6. F7 is acombination of EPA FFA and lower molecular weight TGs based on the FFAmeasurement of 23.1% versus 68.5% for F6. F7 has significant EPA withinit, so this is included with the higher MW fraction despite the presenceof lower MW TGs. Collectively, this data implies that if a FFA feedstockwere extracted with process conditions similar to F3, the resultantextract was concentrated in lower molecular weight FA while the highermolecular weight FA would be the residue. This can be accomplished in acountercurrent column extractor. The extract is the concentrate of thelower molecular weight compounds. The raffinate (column bottom) is theconcentrate of the higher molecular weight compounds, including EPA.Based on the ratio of the recovered mass in each molecular weight class,an effective mass fraction can be defined by molecular weight range, asis given in Table 21. This table shows that 85 wt % of the EPA in thehydrolyzed feedstock can be recovered in the EPA concentrated fraction(raffinate). FIG. 13 shows the distribution of several characteristicmolecular weight components and the FFA level associated with eachfraction.

TABLE 18 Fatty Acid Distribution of Hydrolyzed S14 Algae Oil FeedstockControl Sample Test QLTS-B-8 Control C#:Dbl. Norm. FA in Fatty Acid BondFA % Mixture % Caprylic  8:0 0.15 0.12 Capric 10:0 0.13 0.10 Lauric 12:00.39 0.31 Myristic 14:0 4.74 3.79 Myristoleic 14:1 0.35 0.28Pentadecanoic 15:0 0.40 0.32 Palmitic 16:0 29.04 23.24 Palmitoleic 16:125.45 20.37 Hexadecadienoic 16:2 0.29 0.23 Hexadecatrienoic 16:3 0.230.18 Heptadecanoic 17:0 0.31 0.25 Stearic 18:0 0.86 0.69 Oleic   18:1ω93.80 3.04 Oleic   18:1ω7 0.74 0.59 Linoleic   18:2ω6 3.02 2.42 Linoleic  18:2ω4 0.00 0.00 Gamma-Linolenic   18:3ω6 0.46 0.37 Alpha-Linolenic(ALA)   18:3ω3 1.09 0.87 Arachidic 20:0 0.24 0.19 Eicosadienoic   20:2ω60.21 0.17 Eicosatrienoic   20:3ω6 0.25 0.20 Arachidonic   20:4ω6 4.103.28 Eicosapentaenoic (EPA)   20:5ω3 15.66 12.53 Other n/a 8.10 6.49Total Fatty Acid 100.00 80.03 Total Omega-3 16.75 13.40 Total Omega-68.04 6.44

TABLE 19 FFA and Mass fraction of Pressure Gradient Fractions of aHigh-FFA S14 Oil Feedstock FFA Level Recovered Fraction Sample ID wt %Mass % Control QLTS-B-8 Control 44.70 F1 QLTS-B-8 F1 72.00 4.7 F2QLTS-B-8 F2 94.90 8.3 F3 QLTS-B-8 F3 94.90 12.0 F4 QLTS-B-8 F4 95.50 7.6F5 QLTS-B-8 F5 85.50 8.0 F6 QLTS-B-8 F6 68.50 5.8 F7 QLTS-B-8 F7 23.104.7 F8 QLTS-B-8 F8 19.70 10.1 F9 QLTS-B-8 F9 3.60 8.0 F10 QLTS-B-8 F102.00 7.6 F11 QLTS-B-8 F11 4.70 13.4 F12 QLTS-B-8 F12 18.40 4.7 RecoveredMass, F1-F7, (% of Feed) 51.09 Recovered Mass, F8-F12, (% of Feed) 43.84Mass Balance (Total % of Feed Recovered) 94.93

TABLE 20 Fatty Acid Profile of Pressure Gradient Fractions F1 through F7Fraction F1 F2 F3 F4 Test QLTS-B-8 QLTS-B-8 QLTS-B-8 QLTS-B-8 F1 F2 F3F4 C#:Dbl. Norm. % FA in Norm. % FA in Norm. % FA in Norm. % FA in FattyAcid Bond FA % Fract FA % Fract FA % Fract FA % Fract Caprylic  8:0 1.441.24 0.13 0.12 0.00 0.00 0.00 0.00 Capric 10:0 0.78 0.67 0.14 0.13 0.000.00 0.00 0.00 Lauric 12:0 1.51 1.30 0.82 0.78 0.12 0.11 0.00 0.00Myristic 14:0 10.17 8.74 11.57 10.94 4.35 4.16 0.90 0.85 Myristoleic14:1 5.20 4.47 0.78 0.73 0.35 0.33 0.06 0.05 Pentadecanoic 15:0 0.600.52 0.69 0.66 0.50 0.48 0.20 0.19 Palmitic 16:0 18.84 16.20 30.01 28.3735.83 34.22 28.84 27.08 Palmitoleic 16:1 20.94 18.00 32.88 31.08 31.1529.74 18.46 17.33 Hexadecadienoic 16:2 0.37 0.32 0.45 0.42 0.49 0.460.27 0.25 Hexadecatrienoic 16:3 0.34 0.29 0.43 0.41 0.35 0.33 0.18 0.16Heptadecanoic 17:0 0.00 0.00 0.13 0.12 0.20 0.19 0.29 0.27 Stearic 18:00.14 0.12 0.13 0.12 0.23 0.22 0.60 0.56 Oleic 18:1ω9 1.11 0.96 1.62 1.533.14 3.00 6.13 5.76 Oleic 18:1ω7 0.30 0.26 0.36 0.34 0.64 0.61 1.29 1.21Linoleic 18:2ω6 1.19 1.02 1.83 1.73 3.53 3.37 6.27 5.89 Linoleic 18:2ω40.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Gamma-Linolenic 18:3ω6 0.22 0.190.35 0.33 0.61 0.59 0.88 0.82 Alpha-Linolenic (ALA) 18:3ω3 1.02 0.880.96 0.91 1.49 1.43 2.54 2.38 Arachidic 20:0 0.00 0.00 0.00 0.00 0.000.00 0.14 0.13 Eicosadienoic 20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.130.12 Eicosatrienoic 20:3ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.16 0.15Arachidonic 20:4ω6 0.79 0.68 0.83 0.79 1.99 1.90 5.85 5.49Eicosapentaenoic (EPA) 20:5ω3 2.72 2.34 3.03 2.87 7.18 6.85 20.43 19.18Other n/a 32.32 27.78 12.83 12.13 7.86 7.51 6.39 6.00 Total Fatty Acid100.00 85.97 100.00 94.54 100.00 95.49 100.00 93.89 Total Omega-3 3.743.22 4.00 3.78 8.67 8.28 22.97 21.56 Total Omega-6 2.19 1.89 3.02 2.856.14 5.86 13.29 12.48 Fraction F5 F6 F7 Test QLTS-B-8 QLTS-B-8 QLTS-B-8F5 F6 F7 C#:Dbl. Norm. % FA in Norm. % FA in Norm. % FA in Fatty AcidBond FA % Fract FA % Fract FA % Fract Caprylic  8:0 0.00 0.00 0.00 0.000.13 0.07 Capric 10:0 0.00 0.00 0.05 0.04 0.19 0.09 Lauric 12:0 0.000.00 0.21 0.15 0.89 0.45 Myristic 14:0 0.29 0.24 1.01 0.72 6.21 3.12Myristoleic 14:1 0.00 0.00 0.00 0.00 0.11 0.05 Pentadecanoic 15:0 0.000.00 0.10 0.07 0.40 0.20 Palmitic 16:0 11.38 9.66 7.76 5.55 27.13 13.63Palmitoleic 16:1 6.08 5.16 5.30 3.79 25.13 12.63 Hexadecadienoic 16:20.07 0.06 0.00 0.00 0.20 0.10 Hexadecatrienoic 16:3 0.06 0.05 0.00 0.000.12 0.06 Heptadecanoic 17:0 0.20 0.17 0.12 0.09 0.15 0.07 Stearic 18:00.83 0.70 0.66 0.47 0.47 0.24 Oleic 18:1ω9 6.24 5.30 3.94 2.82 2.94 1.48Oleic 18:1ω7 1.38 1.17 0.88 0.63 0.58 0.29 Linoleic 18:2ω6 5.89 5.003.47 2.48 2.40 1.20 Linoleic 18:2ω4 0.00 0.00 0.00 0.00 0.00 0.00Gamma-Linolenic 18:3ω6 0.65 0.55 0.36 0.26 0.31 0.16 Alpha-Linolenic(ALA) 18:3ω3 2.51 2.13 1.63 1.17 1.20 0.60 Arachidic 20:0 0.66 0.56 1.280.92 0.53 0.26 Eicosadienoic 20:2ω6 0.00 0.00 0.00 0.00 0.00 0.00Eicosatrienoic 20:3ω6 0.59 0.50 0.71 0.51 0.22 0.11 Arachidonic 20:4ω612.15 10.32 12.79 9.16 4.11 2.06 Eicosapentaenoic (EPA) 20:5ω3 43.9637.33 48.85 34.97 18.68 9.39 Other n/a 7.08 6.01 10.78 7.72 7.91 3.97Total Fatty Acid 100.00 84.92 99.90 71.51 100.00 50.24 Total Omega-346.47 39.46 50.48 36.13 19.88 9.99 Total Omega-6 19.27 16.36 17.33 12.417.03 3.53

TABLE 21 Mass Fraction of Major Fatty Acid Components, including EPAConcentration Factor Fraction F4-F7 Lower MW Higher MW Extract RaffinateC#:Dbl. Mass Fraction Mass Fraction Fatty Acid Bond of Feed wt % of Feedwt % Myristic 14:0   86.98 13.02 Palmitic 16:0   65.55 34.45 Palmitoleic16:1   73.32 26.68 Oleic 18:1ω9 32.70 67.30 Oleic 18:1ω7 32.52 67.48Linoleic 18:2ω6 36.26 63.74 Gamma-Linolenic 18:3ω6 45.28 54.72Alpha-Linolenic (ALA) 18:3ω3 39.16 60.84 Arachidic 20:0   14.82 85.18Eicosapentaenoic 20:5ω3 14.48 85.52 (EPA) Other n/a 68.98 31.02 % ofFeed 48.94 51.06

A typical example of EPA concentration is shown in Table 22. A feedstockderived from S12 algae was first fractionated with SCCO2 to remove theNL from other CAO constituents. This mixture was then hydrolyzed to formfree fatty acids. The feed mixture was over 85% FFA. In the feed, theEPA constitutes 46% of the fatty acid and 28.7 wt % of the mixture. Thismixture was concentrated with SCCO2 using the previously describedmethod. In the concentrated EPA mixture, the EPA is 65.1% of the fattyacid and 48.1 wt % of the mixture. There was zero polar lipids in eitherthe FFA feedstock or the EPA concentrate. The ratio of EPA to totalOmega-3 as greater than 99% for both the feedstock material and the EPAconcentrate, a typical value for S12 algae.

This high EPA faction is used to maintain a consistent EPA level in thestandardized formulation. This FFA is in an FFA form, and, thus,facilitates more rapid bioabsorbance. The EPA to total Omega-3 ratio isgreater than 99% in this example. This is used to maintain the highfraction of EPA to total Omega-3 in the standardized formulation. Withrespect to the this blend, the EPA to total EPA ratio is always greaterthan 94% and more typically 95%, 96%, 97%, or 98%.

TABLE 22 Composition of S12 Derived FFA and Concentrated EPA Mixture S12FFA EPA Feedstock Concentrate Norm. FA in Norm. FA in C#:Dbl. FA ExtractFA Extract Fatty Acid Bond % % % % Caprylic  8:0 0.43 0.27 0.09 0.07Capric 10:0 0.37 0.23 0.08 0.06 Lauric 12:0 1.49 0.93 0.32 0.24 Myristic14:0 3.16 1.97 0.68 0.50 Myristoleic 14:1 0.46 0.29 0.10 0.07Pentadecanoic 15:0 0.14 0.09 0.03 0.02 Palmitic 16:0 7.45 4.65 4.25 3.14Palmitoleic 16:1 11.83 7.38 5.22 3.86 Hexadecadienoic 16:2 0.22 0.140.10 0.07 Hexadecatrienoic 16:3 0.32 0.20 0.14 0.11 Stearic 18:0 0.540.34 0.61 0.45 Oleic   18:1ω9 1.73 1.08 1.93 1.42 Oleic   18:1ω7 0.850.53 0.95 0.70 Linoleic   18:2ω6 2.74 1.71 2.89 2.13 Linoleic   18:2ω40.14 0.09 0.15 0.11 Gamma-Linolenic   18:3ω6 0.38 0.24 0.35 0.26Alpha-Linolenic   18:3ω3 0.26 0.16 0.26 0.19 (ALA) Eicosatrienoic  20:3ω6 0.24 0.15 0.34 0.25 Arachidonic   20:4ω6 6.12 3.82 8.63 6.37Eicosapentaenoic   20:5ω3 46.02 28.71 65.13 48.09 (EPA) Other n/a 15.089.41 7.74 5.72 Total Fatty Acid 100.00 62.39 100.00 73.83 Total Omega-346.27 28.87 65.39 48.28 EPA/Omega-3 99.4 99.6 Total Omega-6 9.49 5.9212.21 9.01

Three components are blended to form a standardized combination of EPAand polar lipids: CAO, Conc PoL, and Conc EPA are used to create astandardized product that controls both the EPA and the polar lipidcontent in the blend. Nominally, the EPA is 25 wt %, the total polarlipids are greater than 15 wt % with more than 5 wt % being PL and morethan 10 wt % being GL.

Tables 23 and 24 show the fatty acid profile and polar lipid profile,respectively, of a typical standardized formulation of polar lipids andEPA. In this example, CAO is not used in the mixture. The polar lipidcontribution to the standardized formulation comes from the Conc PoLfraction. The PoL fraction is comprised of phospholipids andglycolipids. In the PoL, the TFA (total fatty acid) can vary between 25and 45 wt. %, where a typical and measured value was about 35 wt. %. Inthis fatty acid, EPA can vary from a low of 5 wt. % to high of 25 wt. %.A typical value was about 10 wt. % (measured). In this particularmeasure, the EPA was about 29 wt. % of the fatty acid distribution.

The ratios of PL and GL can vary. We have purified a PL/GL fraction thatcompletely removes the TG/DG/MG and FFA. Hence, the fraction has zeroneutral lipid. In an example of polar lipid distribution, the PL wasabout 20 wt % and the GL was about 35 wt %. Thus, PL and GL were 37 wt %and 63 wt % of the polar lipids, respectively. Fatty acid wasdistributed between PL and GL as 39.5% and 60.5%, respectively. Giventhat the distribution of fatty acids between the lipid classes is nearlyidentical to the ratio of the two lipid classes, it is most likely thatEPA is distributed uniformly by weight. Thus, a typical EPA distributionwould be 39.5 wt % with the PL and 60.5% with the GL. A reasonable ratioof EPA distribution between PL and GL would be between 3:1 and 1:3.Thus, at the one extreme, EPA can be 64% with the PL and 36% with theGL. At the other extreme, EPA can be 16% with the PL and 83.6% with theGL. It is more likely that the EPA is biased toward the GL than the PL;however, depending on the metabolic and environmental history, the algaecould produce it in either distribution.

The EPA level can be adjusted using the EPA-FFA from the Conc EPA. Notethat the ratio of EPA to total Omega-3 is greater than 99%. The EPAconstitutes greater than 25% of the mixture. A typical value is about25%; however, with more refinement of the EPA concentrate, this valuecould be as much as about 50%. Values characteristic of the standardizedformulation are 30%, 35%, 40%, 45%, and 50%. C16:0 and C16:1,collectively, represent 11% of the mixture. A typical range is 2 to 15wt %. In any event, the total of the C16 fatty acids will be present inthe mixture at an amount greater than 2 wt % and less than 20 wt %.Lower molecular weight compounds, such as C10:0, C12:0, C14:0, arerelatively minor components in the mixture. All these constituents maybe detectable. C14:0 fatty acids are present at greater than 0.2 wt %and less than 5 wt % of the standardized mixture. The C18 compoundsrepresent a minor component of the standardize mixture and are typicallyless than 5% of the composition. The composition contains detectablequantities of C18:0, C18:1ω9, C18:1ω7, C18:2ω6, C18:3ω3 and allcompounds in this list, other than C18:0, are present in thestandardized mixture at a mass fractions greater than 0.2 wt % and lessthan 3 wt %.

TABLE 23 Fatty Acid Composition of Standardized Mixture derived from S12PoL and EPA Concentrate S12 PoL S12 EPA Standardized ConcentrateConcentrate Mixture wt % of Blend 64.7 35.3 C#:Dbl. Norm. % FA in Norm.% FA in Norm. % FA in Fatty Acid Bond FA % Mixture FA % Mixture FA %Mixture Capric 10:0 0.19 0.08 0.08 0.06 0.13 0.07 Lauric 12:0 0.33 0.130.33 0.24 0.33 0.17 Myristic 14:0 3.58 1.43 0.68 0.50 2.12 1.10 Palmitic16:0 14.86 5.93 4.25 3.14 9.53 4.95 Palmitoleic 16:1 18.05 7.21 5.233.86 11.61 6.03 Hexadecadienoic 16:2 0.36 0.14 0.09 0.07 0.23 0.12Stearic 18:0 0.16 0.06 0.61 0.45 0.39 0.20 Oleic 18:1ω9 2.66 1.06 1.921.42 2.29 1.19 Oleic 18:1ω7 0.84 0.33 0.95 0.70 0.89 0.46 Linoleic18:2ω6 4.39 1.75 2.89 2.13 3.64 1.89 Alpha-Linolenic (ALA) 18:3ω3 0.380.15 0.35 0.26 0.36 0.19 Arachidonic 20:4ω6 3.44 1.37 8.63 6.37 6.043.14 Eicosapentaenoic (EPA) 20:5ω3 32.08 12.81 65.14 48.09 48.67 25.26Other n/a 18.68 7.46 8.86 6.54 13.75 7.14 Total Fatty Acid 100.00 39.93100.00 73.83 100.00 51.90 Total Omega-3 32.45 12.96 65.49 48.35 49.0425.45 EPA/Omega-3 98.8 99.5 99.3 Total Omega-6 7.83 3.13 11.51 8.50 9.685.02

TABLE 24 Polar Lipid and Phytonutrient Profile of Standardized Mixturederived from S12 PoL and EPA Concentrate S12 PoL S12 EPA StandardizedConcentrate Concentrate Mixture Class Relative Extract Relative ExtractRelative Extract Component Name Code Basis % Basis % Basis % Basis %Basis % Basis % Phospholipid Phosphatidylcholine PC 45.1 7.42 0.00 45.14.80 Lyso-Phosphatidylcholine 2-LPC 0.0 0.00 0.00 0.0 0.00Phosphatidylinositol PI 0.0 0.00 0.00 0.0 0.00 PhosphatidylethanolaminePE 0.0 0.00 0.00 0.0 0.00 Phosphatidylglycerol PG 25.1 4.13 0.00 25.12.67 Other 29.8 4.90 0.00 29.8 3.17 GlycolipidDigalactosyldiacylglycerol DGDG 72.1 21.97 0.00 72.1 14.22Monogalactosyldiacylglycerol MGDG 27.9 8.50 0.00 27.9 5.50Phytonutrients Phytosterols 0.35 0.85 0.53 Chlorophyll 16.50 1.20 11.10Other Constituents Mannitol 2.97 0.00 1.92 Free Glycerol 0.20 0.00 0.13Total Phospholipids (PL) (wt %) 16.45 0.00 10.64 Total Glycolipids (GL)(wt %) 30.47 0.00 19.72 Total PoL (PL + GL) (wt %) 46.92 0.00 30.36

In Table 24, the polar lipid and phytonutrient composition is shown. ThePL and GL are associated with the PoL concentrate. There is zero PL andGL in the EPA concentrate. Phytosterols have a higher concentration inthe EPA concentrate. This is characteristic of SCCO2 fractionatedmaterial, as carotenoids are highly soluble in SCCO2. The standardizedmixture contains a total of 10.6 wt % total PL and 19.7 wt % total GL.

These are typical values. The PL constituents always contain PC and PG.Other PLs that can exist in the mixture are 2 LPC, PI, and PE. PC and PGis typically greater than 30 and 15 wt %, respectively, of the PLconstituents. The ratio of GL to PL can vary from 0.75 to 4.0, withtypical values being in the range from 1.5 to 2.5. The GL alwayscontains DGDG and MGDG. Typically, DGDG is greater than 50 wt % of theGL with more typical values of 50, 55, 60, 65, 70, 75, or 80 wt %. Thestandardized mixture contains at least 0.1 wt % phytosterols, with atypical range between 0.25 and 0.75 wt %. Chlorophyll is present inlarger quantities. A value of 11.1 wt % is typical. Chlorophyll levelsare no less than 1 wt % of the standardized mixture and more typically5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 wt % of the mixture.Furthermore, the chlorophyll would most commonly be in the range from 8to 12 wt %. The amount of mannitol in the blend is between 0.1 and 3.0wt %. A typical value is around 2 wt % and more typically 0.5, 1.0, 1.5,or 2.0 wt %.

The constituents of a typical embodiment of the standardized mixture areshown in FIG. 14. The formulation includes PL, GL, NL as TG and DG, andNL as FFA. EPA is distributed between all of these lipid classes. Themixture contains minor components of phytonutrients, includingchlorophyll, mannitol, phytosterols, and carotenoids. The distributionof fatty acids among the different lipid classes is shown in FIG. 15,with 53% as FFA, 9% as Neutral TG and DG, 16% as PL, and 22% as GL. TheFFA fraction could be as low as 30% and as high as 60%, with moretypical values being 40, 45, 50, and 55%. Type NL as TG/DG fractioncould be as low as 1 wt % and as high as 14%. Typical values are 5, 6,7, 8, 9, and 10%. PL could vary in the range from 5% to 30%, withtypical values being 12, 14, 16, 18, and 20%. GL could vary on the rangefrom 10% to 40%, with typical values of 16, 18, 20, 22, 24, and 26%.

Example 2 Streamlined Processing Method

This example summarizes a streamlined method for producing the presentEPA formulations that takes advantage of the improvements in thecultivation of Nannochloropsis biomass that raises the EPA fraction inthe fatty acid profile of the algal biomass to greater than 35%. Underthis scenario both the polar lipids and the neutral lipids have enhancedEPA content. A schematic of the methodology is depicted in FIG. 6.Generally, this process eliminates the step of subjecting the CAO toSCCO2 extraction to split the CAO into a neutral lipid fraction and apolar lipid fraction. Instead, a first portion of the CAO is subject tohydrolysis and free fatty acid fractionation and the second portion ofthe CAO is included directly in the final blended EPA formulation.Generally, the first portion and the second portion of the CAO are aboutequal, or have a volume ratio in the range of about 3:1 to about 1:3.

As described in Example 1, an organic solvent comprised from the generaof ketones and alcohols and mixtures thereof, is used to create CAE fromthe wet algal biomass. The solid content of this wet biomass is greaterthan 17 wt. %.

After recovery from the solvent, the resultant CAE can be solubilized inmethanol and then added to a liquid-liquid partitioning system. Atypical solvent combination would be heptane (Hep), ethyl acetate(EtAc), methanol (MeOH), and water (H₂O) in the volume ratio of 1:1:1:1.The mixture is agitated and allowed to settle. The material splits intoan upper organic layer dominated by the Hep and EtAc and lower aqueouslayer comprised of MeOH and H₂O. The neutral and polar lipids, sterols,and cholesterol have a much higher distribution coefficient for theorganic layer and predominantly remain in the organic layer.Water-soluble carbohydrates including mannitol, water-soluble proteins,and glycerol predominantly go into solution within the aqueous layer.

In varying embodiments liquid-liquid partitioning can employ alternateenvironmentally friendly organic solvents for any one of the Hep, EtAc,MeOH, or H₂O components. Illustrative environmentally friendly solventsinclude without limitation water, acetone, ethanol, 2-propanol,1-propanol, ethyl acetate, isopropyl acetate, methanol, methyl ethylketone (MEK), 1-butanol, and t-butanol. Other solvents of use forliquid-liquid partitioning include liquid of cyclohexane, heptane,toluene, methylcylcohexane, methyl t-butyl ether, isooctane,acetonitrile, 2-methyltetrahydrofuran, tetrahydrofuran (THF), xylenes,dimethyl sulfoxide (DMSO), acetic acid, and ethylene glycol.

As a typical example of liquid partitioning, 11.9 g of CAE was dissolvedin 33 mL of MeOH. This solution was then added to a mixture of 125 mL ofEtAc and 125 mL of Hep. The combination was mixed well in a separationfunnel. 125 mL of H₂O was added to this mixture and further agitated.The system was allowed to settle into two phases: an upper organic layerand a lower aqueous layer.

The composition of the feed material was a typical CAE:

Moisture: 1.40 wt %

FFA=5 wt %

Total Fatty Acid (TFA): 36.4 wt %

EPA in mixture: 14.6 wt %

EPA in fatty acid (FA): 40.8%

Total PL: 9.1 wt %

Total GL: 14.9 wt %

Total Polar Lipids (PoL): 24.0 wt %

Cholesterol/phytosterols: 1.0 wt %

Chlorophyll: 11.8 wt %

Mannitol: 7.3 wt %

Glycerol 0.4 wt %

After recovery of the solvent, the organic phase contained the recoveredCAO. This was 66.2 wt % of the feed materials. It had the followingsolvent-free constituents:

Moisture: less than 1.76 wt %

FFA=21.6 wt %

TFA: 51.6 wt %

EPA: 18.5 wt %

EPA in FA: 35.9%

Total PL: 14.3 wt %

Total GL: 21.9 wt %

Total PoL: 36.2 wt %

Phytosterols: 5.4 wt %

Chlorophyll: 5.4 wt %

Mannitol: 0.0 wt %

Glycerol 0.0 wt %

The recovery of the EPA in the organic layer was 95.9%. The EPA contentin this CAO is greater than 15 wt % and total PoL is greater than 25 wt%. Because the feed material has a high EPA content, this CAO can serve,without further processing, as concentrated PoL for forming theStandardized EPA blend.

After evaporation of solvent (including water), the aqueous layer (41.3%of feed) had the following constituents:

Moisture: greater than 1.03 wt %

FFA=32.0 wt %

TFA: 3.14 wt %

EPA: 1.27 wt %

EPA in FA: 40.6%

Total PL: 2.3 wt %

Total GL: 2.0 wt %

Total PoL: 4.3 wt %

Phytosterols: 0.02 wt %

Chlorophyll: 0.3 wt %

Mannitol: 5.8 wt %

Glycerol 2.2 wt %

There was 4.1% loss of EPA into the aqueous layer.

With this CAO, we have the option to either hydrolyze the CAO directly(as shown in FIG. 6) or to hydrolyze a portion of the CAE. In eithercase, all hydrolyzed lipid classes are converted into FFA. This FFA isthen pressure profile fractionated to preferentially remove a high-EPAfraction. This is concentrated EPA FFA (or EPA-FFA). Knowing bothcomposition of the CAO and the EPA-FFA, the mass ratios can bedetermined to create a standardized EPA formulation.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An EPA composition comprising from about 15 wt. %to about 90 wt. % eicosapentaenoic acid (EPA) and about 10 wt. % toabout 70 wt. % polar lipids, wherein: the polar lipids comprisephospholipid conjugates and glycolipid conjugates; about 3 wt. % toabout 50 wt. % of the EPA in the composition is a phospholipidconjugate; about 5 wt. % to about 50 wt. % of the EPA in the compositionis a glycolipid conjugate; about 0 wt. % to about 10 wt. % of the EPA isa triglyceride conjugate or a diglyceride conjugate; the compositioncomprises between about 0.1 wt. % and about 3.0 wt. % mannitol and thecomposition does not comprise docosahexaenoic acid (DHA), fatty acidmethyl esters or fatty acid ethyl esters, and the composition issuitable for human consumption.
 2. The composition of claim 1, whereinabout 15 wt. % to about 85 wt. % of the EPA in the composition is infree fatty acid form.
 3. An EPA composition comprising followingdistribution of EPA by lipid class: about 3 wt. % to about 50 wt. % ofthe EPA is a phospholipid conjugate; about 5 wt. % to about 50 wt. % ofthe EPA is a glycolipid conjugate; about 0 wt. % to about 10 wt. % ofthe EPA is a triglyceride conjugate or a diglyceride conjugate; andabout 15 wt. % to about 85 wt. % of the EPA is in free fatty acid form,wherein the composition does not comprise fatty acid methyl esters orfatty acid ethyl esters, and wherein the composition comprises betweenabout 0.1 wt. % and about 3.0 wt. % mannitol and is suitable for humanconsumption.
 4. The composition of claim 1, wherein the EPA to totalomega-3 fatty acids ratio is greater than 90%.
 5. The composition ofclaim 1, wherein the composition comprises about 15 wt. % to about 75wt. % EPA.
 6. The composition of claim 1, wherein the compositioncomprises about 20 wt. % to about 50 wt. % EPA.
 7. The composition ofclaim 1, wherein the composition comprises at least about 13 wt. % polarlipids.
 8. The composition of claim 1, wherein the composition comprisesat least about 13 wt. % polar lipids, less than 0.2 wt. % glycerideconjugates and at least about 30 wt. % free fatty acids.
 9. Thecomposition of claim 1, wherein the polar lipids are comprised ofphospholipid conjugates and glycolipid conjugates at a wt. % ratio inthe range of about 3:1 to about 1:3.
 10. The composition of claim 9,wherein the glycolipid conjugates comprise one or more ofdigalactosyldiacylglycerol and monogalactosyldiacylglycerol.
 11. Thecomposition of claim 9, wherein the phospholipid conjugates comprise oneor more of phosphatidylcholine, lyso-phosphatidylcholine,phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol.12. The composition of claim 11, wherein the phospholipid conjugatescomprise one or more of phosphatidylcholine and phosphatidylglycerol.13. The composition of claim 1, wherein the composition comprises: i) 0to 5 wt. % C:18 fatty acids; ii) 0 to 20 wt. % C:16 fatty acids; iii) 0to 5 wt. % C:14 fatty acids; iv) 0 to 0.5 wt. % C:12 fatty acids; and/orv) 0 to 0.5 wt. % C:10 fatty acids.
 14. The composition of claim 1,wherein the composition comprises chlorophyll a.
 15. The composition ofclaim 1, wherein the composition comprises less than about 10.0 wt. %arachidonic acid.
 16. The composition of claim 1, wherein thecomposition is substantially free of intact cells, cellular components,polynucleotides, and polypeptides.
 17. The composition of claim 1,wherein the composition comprises less than about 1 wt. % phytosterols.18. The composition of claim 1, wherein the composition comprises lessthan about 2 wt. % carotenoids.
 19. The composition of claim 1, whereinthe composition does not comprise fatty acids selected from the groupconsisting of octadecatetraenoic acid or stearidonic acid (SDA=C18:4ω3),eicosatrienoic acid (ETE=C20:3ω3), eicosatetraenoic acid (ETA=C20:4ω3),heneicosapentaenoic acid or uncosapentaenoic acid (HPA=C21:5ω3), anddocapentaenoic acid (DPA=C22:5ω3).
 20. The composition of claim 1,wherein the composition does not comprise one or more carotenoidsselected from the group consisting of astaxanthin, cis-lutein,trans-lutein, cis-zeaxanthin, trans-alpha-crytoxanthin,trans-alpha-carotene, cis-alpha-carotene, cis-lycopene, andtrans-lycopene.
 21. The composition of claim 1, wherein the compositiondoes not comprise chlorophyll c.
 22. The composition of claim 1, whereinthe composition does not comprise one or more phospholipids selectedfrom the group consisting of N-acyl-phosphatidylethanolamine,lyso-phosphatidylcholine, phosphatidylinositol andphosphatidylethanolamine.
 23. The composition of claim 1, wherein thecomposition comprises about 20-50 wt. % EPA, about 10-25 wt. %glycolipids, and about 5-25 wt. % phospholipids.
 24. The composition ofclaim 1, wherein the composition comprises: Component wt. % Fatty AcidsCapric (10:0) 0.1-0.2 Lauric (12:0) 0.1-0.2 Myristic (14:0) 1.0 Palmitic(16:0) 5.3-6.5 Palmitoleic (16:1) 7.0-8.0 Hexadecadienoic (16:2) 0.2-0.3Stearic (18:0) 0.2-0.3 Oleic (18:1ω9) 2.0 Oleic (18:1ω7) 0.3-0.4Linoleic (18:2ω6) 1.2-2.0 Alpha-Linolenic (ALA) (18:3ω3) 0.2 Arachidonic(20:4ω6) 1.0-8.0 Eicosapentaenoic (EPA) (20:5ω3) 19-30 Total Fatty Acid40-55 Total Omega-3 20-30 EPA/Omega-3 >93    Total Omega-6  2-10Phospholipids Phosphatidylcholine 4.7-7.4 Lyso-Phosphatidylcholine0.3-0.4 Phosphatidylinositol 0.8-1.3 Phosphatidylethanolamine 0.5-0.8Phosphatidylglycerol 1.8-2.8 Glycolipids Digalactosyldiacylglycerol10-17 Monogalactosyldiacylglycerol 3-5 Phytonutrients Phytosterols 1.5Chlorophyll 4-5 Total Phospholipids (PL) (wt %)  9-14 Total Glycolipids(GL) (wt %) 13-21 Total PoL (PL + GL) (wt %)  22-35.


25. A capsule, tablet, solution, syrup, or suspension suitable for humanconsumption comprising a composition of claim
 1. 26. The capsule ofclaim 25, wherein the capsule is a soft capsule.
 27. A food, beverage,energy bar, or nutritional supplement comprising a composition of claim1.